Aminotetralin compounds as mu opioid receptor antagonists

ABSTRACT

The invention provides aminotetralin compounds of formula (I): 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , n, and m are defined in the specification, or a pharmaceutically-acceptable salt thereof, that are antagonists at the mu opioid receptor. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat conditions associated with mu opioid receptor activity, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/007,127 filed on Dec. 11, 2007, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to aminotetralin compounds which are useful asmu opioid receptor antagonists. The invention is also directed topharmaceutical compositions comprising such compounds, methods of usingsuch compounds for treating or ameliorating medical conditions mediatedby mu opioid receptor activity, and processes and intermediates usefulfor preparing such compounds.

2. State of the Art

It is now generally understood that endogenous opioids play a complexrole in gastrointestinal physiology. Opioid receptors are expressedthroughout the body, both in the central nervous system and inperipheral regions including the gastrointestinal (GI) tract.

Compounds which function as agonists at opioid receptors, of whichmorphine is a prototypical example, are the mainstays of analgesictherapy for the treatment of moderate to severe pain. Unfortunately, useof opioid analgesics is often associated with adverse effects on the GItract, collectively termed opioid-induced bowel dysfunction (OBD). OBDincludes symptoms such as constipation, decreased gastric emptying,abdominal pain and discomfort, bloating, nausea, and gastroesophagealreflux. Both central and peripheral opioid receptors are likely involvedin the slowdown of gastrointestinal transit after opioid use. However,evidence suggests that peripheral opioid receptors in the GI tract areprimarily responsible for the adverse effects of opioids on GI function.

Since the side effects of opioids are predominantly mediated byperipheral receptors, whereas the analgesia is central in origin, aperipherally selective antagonist can potentially block undesirableGI-related side effects without interfering with the beneficial centraleffects of analgesia or precipitating central nervous system withdrawalsymptoms.

Of the three major opioid receptor subtypes, denoted mu, delta, andkappa, most clinically-used opioid analgesics are thought to act via muopioid receptor activation to exert analgesia and to alter GI motility.Accordingly, peripherally selective mu opioid antagonists are expectedto be useful for treating opioid-induced bowel dysfunction. Preferredagents will demonstrate significant binding to mu opioid receptors invitro and be active in vivo in GI animal models.

Postoperative ileus (POI) is a disorder of reduced motility of the GItract that occurs after abdominal or other surgery. The symptoms of POIare similar to those of OBD. Furthermore, since surgical patients areoften treated during and after surgery with opioid analgesics, theduration of POI may be compounded by the reduced GI motility associatedwith opioid use. Mu opioid antagonists useful for treating OBD aretherefore also expected to be beneficial in the treatment of POI.

SUMMARY OF THE INVENTION

The invention provides novel compounds that possess mu opioid receptorantagonist activity and intermediates for the preparation thereof.

Accordingly, the invention provides a compound of formula (I):

wherein

R¹ is —OR^(a) or —C(O)NR^(b)R^(c);

R², R³, and R⁴ are each independently C₁₋₃alkyl;

R⁵ and R⁶ are each independently selected from hydrogen, benzyl, andphenyl, wherein phenyl is optionally substituted with halo, or R⁵ and R⁶together with the carbon atom to which they are attached form acyclopentyl or cyclohexyl ring,

R⁷ is selected from hydroxy, C₁₋₃alkoxy, and —NR⁸R⁹;

R⁸ and R⁹ are each independently selected from hydrogen, C₁₋₆alkyl,cyclohexyl, and benzyl, or R⁸ and R⁹ together with the nitrogen atom towhich they are attached form piperidinyl or piperazinyl, whereinpiperidinyl and piperazinyl are optionally substituted with methyl;

R^(a), R^(b), and R^(c) are each independently hydrogen or C₁₋₃alkyl;

n is 0, 1, 2, 3, or 4; and

m is 0 or 1;

wherein the substituents at the chiral centers marked by asterisks arein the trans configuration;

provided that when n+m=1 and R⁷ is hydroxy or C₁₋₃alkoxy, then R⁵ and R⁶are each independently selected from benzyl and phenyl, or R⁵ and R⁶together with the carbon atom to which they are attached form acyclohexyl ring;

or a pharmaceutically-acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier.

The invention also provides a method of treating a disease or conditionassociated with mu opioid receptor activity, e.g. a disorder of reducedmotility of the gastrointestinal tract such as opioid-induced boweldysfunction and post-operative ileus, the method comprisingadministering to the mammal, a therapeutically effective amount of acompound or of a pharmaceutical composition of the invention.

The compounds of the invention can also be used as research tools, i.e.to study biological systems or samples, or for studying the activity ofother chemical compounds. Accordingly, in another of its method aspects,the invention provides a method of using a compound of formula (I), or apharmaceutically acceptable salt thereof, as a research tool forstudying a biological system or sample or for discovering new compoundshaving mu opioid receptor activity, the method comprising contacting abiological system or sample with a compound of the invention anddetermining the effects caused by the compound on the biological systemor sample.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating a disease or condition associated with mu opioid receptoractivity, e.g. a disorder of reduced motility of the gastrointestinaltract, in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides aminotetralin mu opioid receptor antagonists offormula (I), pharmaceutically-acceptable salts thereof, andintermediates for the preparation thereof. The following substituentsand values are intended to provide representative examples of variousaspects of this invention. These representative values are intended tofurther define such aspects and are not intended to exclude other valuesor limit the scope of the invention.

In a specific aspect, R′ is —OR^(a) or —C(O)NR^(b)R^(c).

In another specific aspect, R¹ is —OH or —C(O)NH₂.

In yet another specific aspect, R¹ is —C(O)NH₂.

In a specific aspect, R², R³, and R⁴ are each independently C₁₋₃alkyl.

In another specific aspect, R² and R³ are each independently methyl orethyl.

In yet other aspects, R² and R³ are each ethyl; or R² and R³ are eachmethyl.

In a specific aspect, R⁴ is methyl.

In a specific aspect, R⁵ and R⁶ are each independently selected fromhydrogen, benzyl, and phenyl, wherein phenyl is optionally substitutedwith halo, or R⁵ and R⁶ together with the carbon atom to which they areattached form a cyclopentyl or cyclohexyl ring.

In another specific aspect, R⁵ and R⁶ are each independently selectedfrom hydrogen, benzyl, and phenyl, or R⁵ and R⁶ together with the carbonatom to which they are attached form a cyclohexyl ring.

In yet other specific aspects, R⁵ and R⁶ are each phenyl; or one of R⁵and R⁶ is hydrogen and the other is benzyl; or R⁵ and R⁶ together withthe carbon atom to which they are attached form a cyclohexyl ring.

In still another specific aspect, R⁵ and R⁶ are each hydrogen.

In a specific aspect, R⁷ is selected from hydroxy, C₁₋₃alkoxy, and—NR⁸R⁹ wherein R⁸ and R⁹ are each independently selected from hydrogen,C₁₋₆alkyl, cyclohexyl, and benzyl, or R⁸ and R⁹ together with thenitrogen atom to which they are attached form piperidinyl orpiperazinyl, wherein piperidinyl and piperazinyl are optionallysubstituted with methyl.

In another specific aspect, R⁷ is selected from hydroxy and C₁₋₃alkoxy.In another specific aspect R⁷ is hydrogen, methyl, or ethyl.

In yet another specific aspect, R⁷ is —NR⁸R⁹ wherein R⁸ and R⁹ are eachindependently selected from hydrogen, C₁₋₆alkyl, cyclohexyl, and benzyl,or R⁸ and R⁹ together with the nitrogen atom to which they are attachedform piperidinyl or piperazinyl, wherein piperidinyl and piperazinyl areoptionally substituted with methyl.

In yet another aspect, R⁷ is NR⁸R⁹ wherein R⁸ and R⁹ are each hydrogen.In still another aspect, R⁷ is —NR⁸R⁹ wherein R⁸ and R⁹ are eachindependently selected from hydrogen, C₁₋₆alkyl, and benzyl or R⁸ and R⁹together with the nitrogen atom to which they are attached formpiperidinyl.

In a specific aspect, n is 0, 1, 2, 3, or 4; and m is 0 or 1.

In another aspect, n is 0, 1, 2, 3, or 4 and m is 0. In yet anotheraspect, n is 1 and m is 0. In another aspect, n is 1 and m is 1. Instill another aspect, n is 0 and m is 0.

In yet another aspect, n and m are both 0, R⁵ and R⁶ are both hydrogen,and R⁷ is —NR⁸R⁹.

As described below, the carboxylic acid of the invention in which n andm are both 0 and R⁵ and R⁶ are both hydrogen, is a useful intermediatefor the preparation of the amides of the invention where R⁷ is —NR⁸R⁹.

The invention further provides the compounds of Examples 1-34 herein.

All of the compounds of the invention are in the trans configurationwith respect to the two chiral centers indicated by asterisks in formula(I):

The compounds may be a pure diastereomer or enantiomer, for example, theisomer where the chiral centers are (S),(S), or a mixture of the (S),(S)and the (R),(R) isomers. Such isomeric mixtures are denoted herein bythe prefix trans.

The chemical naming convention used herein is illustrated for thecompound of Example 1

which istrans-6-methoxy-8,8-dimethyl-7-(3-oxo-3-piperidin-1-yl-propylamino)-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide according to the IUPAC conventions as implemented in AutoNomsoftware, (MDL Information Systems, GmbH, Frankfurt, Germany) with theaddition of the trans prefix. For convenience, the bicyclic1,2,3,4-tetrahydronaphthalen-2-ylamino group is alternatively referredto herein, by the common name, “aminotetralin”.

In addition to the stereochemistry of the aminotetralin group, thecompounds of the invention may contain a chiral center at the carbonatom to which the substituents R⁵ and R⁶ are attached. Accordingly, theinvention includes pure diastereomers or enantiomers, mixtures ofdiastereomers, racemic mixtures, and stereoisomer-enriched mixtures ofisomers, unless otherwise indicated. When the stereochemistry of acompound is specified, it will be understood by those skilled in theart, that minor amounts of other stereoisomers may be present in thecompositions of the invention unless otherwise indicated, provided thatany utility of the composition as a whole is not eliminated by thepresence of such other isomers.

DEFINITIONS

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), sec-butyl, isobutyl,tert-butyl, n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl,3-methylbutyl, 2-ethylbutyl, 2,2-dimethylpentyl, 2-propylpentyl, and thelike.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “compound” means a compound that was synthetically prepared orprepared in any other way, such as by in vivo metabolism.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes one or more of the following:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient, including        counteracting the effects of other therapeutic agents;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

The term “pharmaceutically-acceptable salt” means a salt prepared froman acid or base which is acceptable for administration to a patient,such as a mammal. Such salts can be derived frompharmaceutically-acceptable inorganic or organic acids and frompharmaceutically-acceptable bases. Typically,pharmaceutically-acceptable salts of compounds of the present inventionare prepared from acids.

Salts derived from pharmaceutically-acceptable acids include, but arenot limited to, acetic, adipic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic,glycolic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, oxalic, pantothenic, phosphoric,succinic, sulfuric, tartaric, p-toluenesulfonic, xinafoic(1-hydroxy-2-naphthoic acid), naphthalene-1,5-disulfonic acid and thelike.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS); andthe like.

The term “hydroxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a hydroxy group. Representativehydroxy-protecting groups include, but are not limited to, alkyl groups,such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoylgroups, such as acetyl; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) andtert-butyldimethylsilyl (TBS); and the like.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.Although a particular aspect of the present invention is illustrated inthe schemes below, those skilled in the art will recognize that allaspects of the present invention can be prepared using the methodsdescribed herein or by using other methods, reagents and startingmaterials known to those skilled in the art. It will also be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group, as well assuitable conditions for protection and deprotection, are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

In one method of synthesis, compounds of the invention of formula (I)may be prepared by a general process shown in Scheme A. (Thesubstituents and variables shown in the following schemes have thedefinitions provided above unless otherwise indicated).

The variable R^(7a) in intermediate (III) represents R⁷ or R^(7a)represents —OP¹ where P¹ is a hydroxy-protecting group, in which casethe process also includes a deprotection step, not shown, to provide acompound of formula (I) in which R⁷ is hydroxy. When R^(7a) isC₁₋₃alkoxy, the product of the reaction may be a mixture of an ester anda carboxylic acid, i.e. of a compound of formula (I) in which R⁷ isC₁₋₃alkoxy and a compound of formula (I) in which R⁷ is hydroxy.

In Scheme A, intermediate (II) is reductively N-alkylated by reactionwith the aldehyde (III). The reaction is typically conducted bycontacting intermediate (II) with between about 1 and about 2equivalents of an aldehyde of formula (III) in a suitable inert diluent,such as dichloromethane, methanol, or 2-methyltetrahydrofuran, in thepresence of between about 1 and about 6 equivalents of a reducing agent,and between about 1 and about 2 equivalents of base, such astriethylamine or N,N-diisopropylethyl amine. The reaction is typicallyconducted at a temperature in the range of about 0° C. to ambienttemperature for about a half hour to about 3 hours or until the reactionis substantially complete. Typical reducing agents include sodiumtriacetoxyborohydride, sodium borohydride, and sodium cyanoborohydride.

In another method of synthesis, compounds of formula (I) in which n andm are both 0 can be prepared by one or more of the reaction steps ofScheme B, depending on the value of the variable R⁷:

where R¹⁰ represents C₁₋₃alkyl. In step (i), addition of the amine ofintermediate (II) to the olefin reagent (IV) provides an ester (V) ofthe invention (R⁷ is C₁₋₃alkoxy). The ester may be converted to thecarboxylic acid (VI) of the invention (R⁷ is hydroxy) (step (ii)),which, in turn may be reacted with an amine HNR⁸R⁹ (VII) to provide theamide (VIII) of the invention (R⁷ is —NR⁸R⁹) (step (iii)).

The Michael addition reaction of step (i) is typically conducted bycontacting aminotetralin intermediate (II) with between about 2 andabout 5 equivalents of intermediate (IV) in a protic diluent, forexample, ethanol, in the presence of between about 1 and about 3equivalents of base, such as triethylamine or N,N-diisopropylethylamine.The reaction is typically conducted at a temperature in the range ofabout 70 to about 100° C. for about 1 to about 4 days or until thereaction is substantially complete. In the reaction of step (ii),treatment of the ester (V) with between about 2 and about 6 equivalentsof base, such as sodium hydroxide provides the carboxylic acid (VI).

Finally, as shown in step (iii), to prepare amides (VIII), the aminereagent (VII) is contacted with between about 2 and about 5 equivalentsof the acid (VI) in an inert diluent, in the presence of base, asdescribed above, and in the presence of between about 3 and about 6equivalents of an activating agent such as N,N-carbonyl diimidazole(CDI), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU) or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC). The reaction istypically conducted at a temperature in the range of about 25° C. toabout 100° C. for about 2 hours to about 24 hours, or until the reactionis substantially complete.

In yet another method of synthesis, amides of the invention in which R⁵and R⁶ are both hydrogen may be prepared as shown in Scheme C:

where L is a halo leaving group such as bromo. First, intermediate (X)is formed by contacting aminotetralin (II) with about 1 equivalent ofthe haloalkylnitrile (IX) in the presence of between about 2 and about 4equivalents of base, for example, sodium carbonate. The nitrile (X) isthen hydrolyzed to form the amide (XI). Alternatively, as available, areagent of the form L-(CH₂)_(q)—C(O)NH₂, where q is n+m+2, for example,6-bromohexanamide can be added directly to intermediate (II) to form(XI).

An exemplary procedure for the preparation of an aminotetralinintermediate (II) in which the variable R¹ is —C(O)NH₂ is illustrated inScheme D

where P¹ represents a hydroxy-protecting group, P² represents anamino-protecting group, and —OTf represents trifluoromethane sulfonate(commonly triflate). The notation “Rac” indicates the compound is aracemic mixture of the particular structure depicted and the structurehaving the opposite stereochemistry at the chiral centers.

A small alkyl is useful as the protecting group P¹. Using an alkyl forP¹, aziridine intermediate 1, can be reacted with HBr to provideintermediate 2 which is conveniently isolated in solid form as the HBrsalt. Typically intermediate 1 is contacted with an excess, for examplebetween about 12 and about 18 equivalents, of HBr. The efficiency of thereaction is improved by the inclusion of a phase transfer catalyst. Thereaction is typically conducted at a temperature between about 90 andabout 110° C. for between about 10 and about 20 hours or until thereaction is substantially complete. Using Boc, for example, for theprotecting group P², intermediate 3 is then formed by treating 2 withbase, which reforms the aziridine ring in situ, and adding between about1 and about 1.3 equivalents of di-text-butyl dicarbonate (commonly(Boc)₂O) under conventional reaction conditions to provide intermediate3.

Alternatively, the P¹ group of aziridine intermediate 1 is deprotectedin two steps by reaction with HBr or BBr₃ and subsequent treatment withbase to provide intermediate 2a:

which is then protected at the aziridine nitrogen, for example byreaction with (Boc)₂O to provide intermediate 3.

Next, the amino-protected aziridine 3 is contacted with a large excessof an alcohol R⁴OH in the presence of a mild acid catalyst, such aspyridium tosylate to provide intermediate 4.

An aminotetralin intermediate of formula (II) in which R¹ is —OH can beprepared by deprotection of intermediate 4. For example, when theprotecting group P² is Boc, the phenol intermediate of formula (II) isobtained by treating 4 with an acid. Similarly, an aminotetralinintermediate of formula (II) in which R¹ is —OR^(a) where R^(a) isC₁₋₃alkyl, can be prepared analogously starting with an intermediate offormula I in which P¹ is the desired small alkyl and omitting theinitial deprotection step.

The remainder of the steps in Scheme D depict the conversion of thehydroxy substituted aminotetralin 4 to a carboxamide substitutedintermediate 7 and a final deprotection step. The hydroxyl ofintermediate 4 is first converted to the triflate by contacting 4 in aninert diluent with between about 1 and about 2 equivalents oftrifluoromethane sulfonylcholoride in the presence of between about 1and about 3 equivalents of base, such as triethylamine to provideintermediate 5. Reaction of 5 with zinc cyanide in the presence of atransition metal catalyst, provides intermediate 6. This reaction istypically conducted at a temperature between about 80° C. and 120° C.under an inert atmosphere for about one half to about 2 hours or untilthe reaction is substantially complete.

Next, the nitrile of intermediate 6 is hydrolyzed to the carboxamide ofintermediate 7. As described in the examples below, in one method ofsynthesis, the nitrile 6 is contacted with between about 5 and about 8equivalents of sodium perborate monohydrate in an inert diluent such asmethanol. The reaction is conducted at a temperature between about 50and about 60° C. for about 12 to about 24 hours or until the reaction issubstantially complete. Alternative processes for hydrolysis of anitrile to an amide include use of a platinum catalyst, in particular,hydrido(dimethylphosphoniousacid-kP)[hydrogenbis(dimethylphosphinito-kP)]platinum(II), and treatment with hydrogenperoxide, as described in examples below. Finally, intermediate 7 isdeprotected by conventional treatment with an acid to provide theaminotetralin of formula (II).

An intermediate of formula (II) in which R¹ is —C(O)NR^(b)R^(c) whereR^(b) and R^(c) are alkyl can be prepared from intermediate 6 byconverting the nitrile to a carboxylic acid by hydrolysis in thepresence of a base followed by amide coupling with an amine of theformula HNR^(b)R^(c).

The individual enantiomers of formula (II) can be separated using achiral auxiliary. Scheme C illustrates use of the chiral auxiliarycarbonic acid 4-nitro-phenyl ester (R)-1-phenyl-ethyl ester (8):

to prepare a pair of non-racemic diastereomers 9a and 9b that can beseparated. The notation “Abs” denotes the specific chiral compoundshown. The racemic aminotetralin (II) is contacted with between about0.8 and about 1.2 equivalents of chiral auxiliary 8 in an inert diluentin the presence of between about 2 and about 4 equivalents of a base,such as triethylamine to prepare a diastereomeric mixture ofintermediates 9a and 9b. The reaction is typically conducted at atemperature between about 80 and about 95° C. for between about 4 andabout 20 hours or until the reaction is substantially complete. Thediastereomers 9a and 9b can be separated by high performance liquidchromatography (HPLC) and collected separately or by crystallization inwhich the diastereomer 9a crystallized preferentially, leavingpredominantly the diastereomer 9b in solution. Finally, the carbamategroup can be removed from the isolated 9a and 9b diastereomers bytreatment with an acid to provide the individual enantiomers ofaminotetralin (II). The chiral auxiliary 8 can be prepared by reactionof (R)-1-phenylethanol with p-nitrophenyl chloroformate as described inthe examples below.

The aziridine intermediate 1 used in Scheme D can be obtained byreacting a substituted 3,4-dihydro-1H-naphthalen-2-one:

with an alkyl halide to add the alkyl substituents R² and R³ at the2-position, treatment with a hydroxylamine salt to convert the carboxyto an oxime and subsequent treatment with lithium aluminum hydride orother reducing agent to convert the oxime to the aziridine 1, asdescribed, for example, in U.S. Pat. No. 6,844,368, and in Preparation12 below.

The aldehyde (III) used in Scheme A is conveniently prepared from thecorresponding carboxylic acid 10 as shown in Scheme F:

Borane reduction of the carboxylic acid 10 provides the alcohol 11. Thereaction is typically conducted by contacting acid 10 with about 2equivalents of a borane-tetrahydrofuran complex in tetrahydrofuran at atemperature between about −5 and about 0° C. The alcohol 11 is thenoxidized to the aldehyde (III) in the presence of oxidizing reagents,for example dimethylsulfoxide with a sulfur trioxide pyridine complexactivating agent. An intermediate 10 used for the preparation ofcompounds of the invention in which R⁵ and R⁶ together with the carbonatom to which they are attached form a cyclohexyl ring is convenientlyobtained from 1,1-cyclohexanediacetic anhydride (See Example 14.)

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereto are described in the examples below.

Accordingly, in a method aspect, the invention provides a process forpreparing a compound of formula (I), or a salt thereof, the processcomprising reacting a compound of formula (II) with (a) a compound offormula (III), and, when R^(7a) is —OP¹, removing the protecting groupP¹, or with (b) a compound of formula (IV) to provide a compound offormula (V), and optionally contacting (V) with base to provide acompound of formula (VI), and optionally reacting (VI) with an amine offormula (VII); or with (c) a compound of formula (IX) to provide acompound of formula (X), and hydrolyzing (X), to provide a compound offormula (I) or a salt thereof.

Pharmaceutical Compositions

The aminotetralin compounds of the invention are typically administeredto a patient in the form of a pharmaceutical composition or formulation.Such pharmaceutical compositions may be administered to the patient byany acceptable route of administration including, but not limited to,oral, rectal, vaginal, nasal, inhaled, topical (including transdermal)and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof. Optionally, such pharmaceutical compositionsmay contain other therapeutic and/or formulating agents if desired. Whendiscussing compositions, the “compound of the invention” may also bereferred to herein as the “active agent”. As used herein, the term“compound of the invention” is intended to include compounds of formula(I), and, in addition, pharmaceutically-acceptable salts and solvates ofthe compound unless otherwise indicated.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically-acceptable salt thereof. Typically, suchpharmaceutical compositions will contain from about 0.1 to about 95% byweight of the active agent; preferably, from about 5 to about 70% byweight; and more preferably from about 10 to about 60% by weight of theactive agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a compound of the presentinvention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methyl cellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions, inaddition to the active ingredient, may contain suspending agents suchas, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compounds of this invention can also be administered parenterally(e.g. by intravenous, subcutaneous, intramuscular or intraperitonealinjection). For parenteral administration, the active agent is typicallyadmixed with a suitable vehicle for parenteral administration including,by way of example, sterile aqueous solutions, saline, low molecularweight alcohols such as propylene glycol, polyethylene glycol, vegetableoils, gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,buffering agents, or dispersing agents. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compounds of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example,the active agent can be admixed with permeation enhancers, such aspropylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-onesand the like, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the compounds of this invention may be administered incombination with one or more other therapeutic agents. In thisembodiment, a compound of this invention is either physically mixed withthe other therapeutic agent to form a composition containing bothagents; or each agent is present in separate and distinct compositionswhich are administered to the patient simultaneously or sequentially inany order.

For example, a compound of formula I can be combined with secondtherapeutic agent using conventional procedures and equipment to form acomposition comprising a compound of formula I and a second therapeuticagent. Additionally, the therapeutic agents may be combined with apharmaceutically acceptable carrier to form a pharmaceutical compositioncomprising a compound of formula I, a second therapeutic agent and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described herein.

Alternatively, the therapeutic agents may remain separate and distinctbefore administration to the patient. In this embodiment, the agents arenot physically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Whenadministered separately, the agents are administered sufficiently closein time so as to provide a desired therapeutic effect. Such compositionscan be packaged separately or may be packaged together as a kit. The twotherapeutic agents in the kit may be administered by the same route ofadministration or by different routes of administration.

In particular, the compounds of the invention can be combined withopioid analgesic therapeutic agents. As described above, use of opioidanalgesics is often associated with undesirable side effects such as,for example, constipation, decreased gastric emptying, abdominal pain,bloating, nausea, and gastroesophageal reflux. These adverse effects maybe sufficiently severe to limit the dose of opioid analgesic that can bedelivered to a patient to a suboptimal level. Coadministration of acompound of the invention with an opioid is likely to reduce or preventside effects thereby increasing the utility of the analgesic agent forpain alleviation.

Opioid analgesics that may be used in combination with compounds of thepresent invention include, but are not limited to, morphine,hydromorphone, oxymorphone, pethidine, codeine, dihydrocodeine,oxycontin, oxycodone, hydrocodone, sufentanil, fentanyl, remifentanil,buprenorphine, butorphanol, tramadol, methadone, heroin, propoxyphene,meperidine, levorphenol, pentazocine, and combinations of opioidanalgesics with ibuprofen or acetaminophen. Compounds of the inventioncould be used in doses ranging from about 0.05 to about 100 mg per dayfor an average 70 kg patient, when combined with an opioid analgesic atits therapeutic dose, for example, when combined with oxycodone at adose of between about 5 mg and about 160 mg per day.

In addition, prokinetic agents acting via mechanisms other than muopioid receptor antagonism may be used in combination with the presentcompounds. For example, 5-HT₄ receptor agonists, such as tegaserod,renzapride, mosapride, prucalopride,1-isopropyl-1H-indazole-3-carboxylic acid{(1S,3R,5R)-8-[2-(4-acetylpiperazin-1-yl)ethyl]-8-azabicyclo[3.2.1]oct-3-yl}amide,1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide,or4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester may be used as the second therapeutic agent.

Additional useful prokinetic agents include, but are not limited to,5-HT₃ receptor agonists (e.g. pumosetrag), 5-HT_(1A) receptorantagonists (e.g. AGI 001), alpha-2-delta ligands (e.g. PD-217014),chloride channel openers (e.g. lubiprostone), dopamine antagonists (e.g.itopride, metaclopramide, domperidone), GABA-B agonists (e.g. baclofen,AGI 006), kappa opioid agonists (e.g. asimadoline), muscarinic M₁ and M₂antagonists (e.g. acotiamide), motilin agonists (e.g. mitemcinal),guanylate cyclase activators (e.g. MD-1100) and ghrelin agonists (e.g.Tzp 101, RC 1139).

Numerous additional examples of such therapeutic agents are known in theart and any such known therapeutic agents may be employed in combinationwith the compounds of this invention. Secondary agent(s), when included,are present in a therapeutically effective amount, i.e. in any amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. Suitable doses for the othertherapeutic agents administered in combination with a compound of theinvention are typically in the range of about 0.05 μg/day to about 100mg/day.

Accordingly, the pharmaceutical compositions of the invention optionallyinclude a second therapeutic agent as described above.

The following examples illustrate representative pharmaceuticalcompositions of the present invention:

Formulation Example A Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), spray-dried lactose (200 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is loaded into a hard gelatin capsule (260 mg of compositionper capsule).

Formulation Example B Hard Gelatin Capsules for Oral Administration

A compound of the invention (20 mg), starch (89 mg), microcrystallinecellulose (89 mg), and magnesium stearate (2 mg) are thoroughly blendedand then passed through a No. 45 mesh U.S. sieve. The resultingcomposition is loaded into a hard gelatin capsule (200 mg of compositionper capsule).

Formulation Example C Gelatin Capsules for Oral Administration

A compound of the invention (10 mg), polyoxyethylene sorbitan monooleate(50 mg), and starch powder (250 mg) are thoroughly blended and thenloaded into a gelatin capsule (310 mg of composition per capsule).

Formulation Example D Tablets for Oral Administration

A compound of the invention (5 mg), starch (50 mg), andmicroscrystalline cellulose (35 mg) are passed through a No. 45 meshU.S. sieve and mixed thoroughly. A solution of polyvinylpyrrolidone (10wt % in water, 4 mg) is mixed with the resulting powders, and thismixture is then passed through a No. 14 mesh U.S. sieve. The granules soproduced are dried at 50-60° C. and passed through a No. 18 mesh U.S.sieve. Sodium carboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg)and talc (1 mg), which have previously been passed through a No. 60 meshU.S. sieve, are then added to the granules. After mixing, the mixture iscompressed on a tablet machine to afford a tablet weighing 100 mg.

Formulation Example E Tablets for Oral Administration

A compound of the invention (25 mg), microcrystalline cellulose (400mg), fumed silicon dioxide (10 mg), and stearic acid (5 mg) arethoroughly blended and then compressed to form tablets (440 mg ofcomposition per tablet).

Formulation Example F Single-scored Tablets For Oral Administration

A compound of the invention (15 mg), cornstarch (50 mg), croscarmellosesodium (25 mg), lactose (120 mg), and magnesium stearate (5 mg) arethoroughly blended and then compressed to form single-scored tablet (215mg of compositions per tablet).

Formulation Example G Suspension for Oral Administration

The following ingredients are thoroughly mixed to form a suspension fororal administration containing 100 mg of active ingredient per 10 mL ofsuspension:

Ingredients Amount Compound of the invention 0.1 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

Formulation Example H Dry Powder Composition

A micronized compound of the invention (1 mg) is blended with lactose(25 mg) and then loaded into a gelatin inhalation cartridge. Thecontents of the cartridge are administered using a powder inhaler.

Formulation Example J Injectable Formulation

A compound of the invention (0.1 g) is blended with 0.1 M sodium citratebuffer solution (15 mL). The pH of the resulting solution is adjusted topH 6 using 1 N aqueous hydrochloric acid or 1 N aqueous sodiumhydroxide. Sterile normal saline in citrate buffer is then added toprovide a total volume of 20 mL.

Formulation Example K Single-Scored Tablets for Oral Administration

A compound of the invention (10 mg), oxycodone hydrochloride (10 mg),cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120 mg), andmagnesium stearate (5 mg) are thoroughly blended and then compressed toform single-scored tablet (220 mg of compositions per tablet).

Formulation Example L Injectable Formulation

A compound of the invention (0.1 g) and oxycodone hydrochloride (0.1 g)are blended with 0.1 M sodium citrate buffer solution (15 mL). The pH ofthe resulting solution is adjusted to pH 6 using 1 N aqueoushydrochloric acid or 1 N aqueous sodium hydroxide. Sterile normal salinein citrate buffer is then added to provide a total volume of 20 mL.

It will be understood that any form of the compounds of the invention,(i.e. free base, pharmaceutical salt, or solvate) that is suitable forthe particular mode of administration, can be used in the pharmaceuticalcompositions discussed above.

Utility

The aminotetralin compounds of the invention are antagonists at the muopioid receptor and therefore are expected to be useful for treatingmedical conditions mediated by mu opioid receptors or associated with muopioid receptor activity, i.e. medical conditions which are amelioratedby treatment with a mu opioid receptor antagonist. In particular, thecompounds of the invention are expected to be useful for treatingadverse effects associated with use of opioid analgesics, i.e. symptomssuch as constipation, decreased gastric emptying, abdominal pain,bloating, nausea, and gastroesophageal reflux, termed collectivelyopioid-induced bowel dysfunction. The mu opioid receptor antagonists ofthe invention are also expected to be useful for treating post-operativeileus, a disorder of reduced motility of the gastrointestinal tract thatoccurs after abdominal or other surgery. In addition, it has beensuggested that mu opioid receptor antagonist compounds may be used forreversing opioid-induced nausea and vomiting. Further, those mu opioidreceptor antagonists exhibiting some central penetration may be usefulin the treatment of dependency on, or addiction to, narcotic drugs,alcohol, or gambling, or in preventing, treating, and/or amelioratingobesity.

Since compounds of the invention increase motility of thegastrointestinal (GI) tract in animal models, the compounds are expectedto be useful for treating disorders of the GI tract caused by reducedmotility in mammals, including humans. Such GI motility disordersinclude, by way of illustration, chronic constipation,constipation-predominant irritable bowel syndrome (C-IBS), diabetic andidiopathic gastroparesis, and functional dyspepsia.

In one aspect, therefore, the invention provides a method of increasingmotility of the gastrointestinal tract in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the invention.

When used to treat disorders of reduced motility of the GI tract orother conditions mediated by mu opioid receptors, the compounds of theinvention will typically be administered orally in a single daily doseor in multiple doses per day, although other forms of administration maybe used. For example, particularly when used to treat post-operativeileus, the compounds of the invention may be administered parenterally.The amount of active agent administered per dose or the total amountadministered per day will typically be determined by a physician, in thelight of the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating disorders of reduced motility of the GItract or other disorders mediated by mu opioid receptors will range fromabout 0.0007 to about 20 mg/kg/day of active agent, including from about0.0007 to about 1.4 mg/kg/day. For an average 70 kg human, this wouldamount to from about 0.05 to about 100 mg per day of active agent.

In one aspect of the invention, the compounds of the invention are usedto treat opioid-induced bowel dysfunction. When used to treatopioid-induced bowel dysfunction, the compounds of the invention willtypically be administered orally in a single daily dose or in multipledoses per day. Preferably, the dose for treating opioid-induced boweldysfunction will range from about 0.05 to about 100 mg per day.

In another aspect of the invention, the compounds of the invention areused to treat post-operative ileus. When used to treat post-operativeileus, the compounds of the invention will typically be administeredorally or intravenously in a single daily dose or in multiple doses perday. Preferably, the dose for treating post-operative ileus will rangefrom about 0.05 to about 100 mg per day.

The invention also provides a method of treating a mammal having adisease or condition associated with mu opioid receptor activity, themethod comprising administering to the mammal a therapeuticallyeffective amount of a compound of the invention or of a pharmaceuticalcomposition comprising a compound of the invention.

As described above, compounds of the invention are mu opioid receptorantagonists. The invention further provides, therefore, a method ofantagonizing a mu opioid receptor in a mammal, the method comprisingadministering a compound of the invention to the mammal.

The mu opioid receptor antagonists of the invention are optionallyadministered in combination with another therapeutic agent or agents, inparticular, in combination with opioid analgesics or with prokineticagents acting via non-mu opioid mechanisms. Accordingly, in anotheraspect, the methods and compositions of the invention further comprise atherapeutically effective amount of an opioid analgesic or anotherprokinetic agent. The methods of the invention include, for example, amethod of reducing or preventing a side effect associated with use of anopioid agent in a mammal, the method comprising administering to themammal an opioid agent and a compound of the invention.

In addition, the compounds of the invention are also useful as researchtools for investigating or studying biological systems or samples havingmu opioid receptors, or for discovering new compounds having mu opioidreceptor activity. Any suitable biological system or sample having muopioid receptors may be employed in such studies which may be conductedeither in vitro or in vivo. Representative biological systems or samplessuitable for such studies include, but are not limited to, cells,cellular extracts, plasma membranes, tissue samples, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, etc.) and the like. Theeffects of contacting a biological system or sample comprising a muopioid receptor with a compound of the invention are determined usingconventional procedures and equipment, such as the radioligand bindingassay and functional assay described herein or other functional assaysknown in the art. Such functional assays include, but are not limitedto, ligand-mediated changes in intracellular cyclic adenosinemonophosphate (cAMP), ligand-mediated changes in activity of the enzymeadenylyl cyclase, ligand-mediated changes in incorporation of analogs ofguanosine triphosphate (GTP), such as [³⁵S]GTPγS (guanosine5′-O-(γ-thio)triphosphate) or GTP-Eu, into isolated membranes viareceptor catalyzed exchange of GTP analogs for GDP analogs, andligand-mediated changes in free intracellular calcium ions. A suitableconcentration of a compound of the invention for such studies typicallyranges from about 1 nanomolar to about 500 nanomolar.

When using compounds of the invention as research tools for discoveringnew compounds have mu opioid receptor activity, binding or functionaldata for a test compound or a group of test compounds is compared to themu opioid receptor binding or functional data for a compound of theinvention to identify test compounds that have superior binding orfunctional activity, if any. This aspect of the invention includes, asseparate embodiments, both the generation of comparison data (using theappropriate assays) and the analysis of the test data to identify testcompounds of interest.

Among other properties, compounds of the invention have been found toexhibit potent binding to mu opioid receptors and little or no agonismin mu receptor functional assays. Therefore, the compounds of theinvention are potent mu opioid receptor antagonists. Further, compoundsof the invention have demonstrated predominantly peripheral activity ascompared with central nervous system activity in animal models.Therefore, these compounds can be expected to reverse opioid-inducedreductions in GI motility without interfering with the beneficialcentral effects of analgesia. These properties, as well as the utilityof the compounds of the invention, can be demonstrated using various invitro and in vivo assays well-known to those skilled in the art.Representative assays are described in further detail in the followingexamples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings

-   -   AcOH=acetic acid    -   ACN=acetonitrile    -   Boc=tert-butoxycarbonyl    -   (Boc)₂O=di-tert-butyl dicarbonate    -   DCM=dichloromethane    -   DIPEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   EtOH=ethanol    -   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   MeOH=methanol    -   MeTHF=2-methyl-tetrahydrofuran    -   RT=room temperature    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Reactionswere run under nitrogen atmosphere, unless noted otherwise. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents.Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR measurement, samples were dissolved indeuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆), and ¹H-NMR spectra wereacquired with a Varian Gemini 2000 instrument (400 MHz) under standardobservation conditions. Mass spectrometric identification of compoundswas performed by an electrospray ionization method (ESMS) with anApplied Biosystems (Foster City, Calif.) model API 150 EX instrument oran Agilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1:7,7-diethyl-5-hydroxy-1a,2,7,7a-tetrahydro-1-aza-cyclopropa[b]naphthalene-1-carboxylicacid tert-butyl ester a.7-amino-6-bromo-8,8-diethyl-5,6,7,8-tetrahydronaphthalen-2-olhydrobromide

To a flask was added7,7-diethyl-5-methoxy-1a,2,7,7a-tetrahydro-1H-1-aza-cyclopropa[b]naphthalene(268 g, 1.16 mol) and hydrogen bromide (1.97 L, 17.38 mol), followed bytetra-N-butylammonium bromide (38 g, 0.12 mol). The reaction mixture washeated at 100° C. overnight with stirring, cooled to room temperatureand then poured into stirred ethyl acetate (2.5 L). The product wasisolated by filtration, the filter cake was washed with ethyl acetate(2×200 mL) and dried to yield crude product (370 g) as a purplish solid.The crude product was suspended in ethanol (1.50 L) then heated at 80°C. for 30 min. The resulting slurry was cooled to room temperature over1 h, and filtered. The flask and filter cake with were washed withethanol (2×100 mL) and then with ethyl acetate (100 mL) and driedovernight to yield the title compound (275 g, ˜96% purity).

b.7,7-diethyl-5-hydroxy-1a,2,7,7a-tetrahydro-1-aza-cyclopropa[b]naphthalene-1-carboxylicacid tert-butyl ester

To a slurry of7-amino-6-bromo-8,8-diethyl-5,6,7,8-tetrahydronaphthalen-2-olhydrobromide (20.0 g) and ethyl acetate (200 mL) was added 1.0 M sodiumhydroxide in water (106 mL). The reaction mixture was stirred at 25° C.for 2 h, di-tert-butyldicarbonate (15 g, 68 mmol) in ethyl acetate (5mL) was added and the reaction mixture was stirred at room temperaturefor 2 h. Following removal of two-thirds of the ethyl acetate (135 mL),heptane (135 mL) was added and the resulting slurry was stirred at roomtemperature over 30 min and then at 5° C. overnight. The slurry wasfiltered, and the filter cake was rinsed with water (100 mL), rinsedwith heptane (50 mL), and dried under vacuum to give the title compound(14.3 g).

Preparation 2:trans-(7-Cyano-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester a.trans-(1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester

To a slurry of7,7-diethyl-5-hydroxy-1a,2,7,7a-tetrahydro-1-aza-cyclopropa[b]naphthalene-1-carboxylicacid tert-butyl ester (170.0 g, 535.6 mmol) and methanol (1700 mL) wasadded pyridinium p-toluenesulfonate (13.4 g, 53.6 mmol) and the reactionmixture was stirred at 40° C. for 4 h. The volume was reduced by rotaryevaporation to ˜300 mL resulting in a thick white slurry. The productwas isolated by filtration; the filter cake was washed with coldmethanol (50 mL) and dried in air for 3 h to yield the title compound(150 g). The filtrate was reduced to ˜50 mL and stirred at 0° C. for 2h, filtered, and dried to yield additional product (25 g).

b. trans-Trifluoro-methanesulfonic acid7-tert-butoxycarbonylamino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ylester

A mixture oftrans-(1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester (195.0 g, 0.558 mol), triethylamine (160 mL, 1.1mol) and ethyl acetate (2000 mL) was stirred at room temperature for 15min and cooled to 0° C. followed by slow addition oftrifluoro-methanesulfonyl chloride (150 g, 0.89 mol) keeping theinternal temperature below 4° C. The resulting slurry was stirred at 0°C. for 1 h. Additional triethylamine (16 mL) followed by additionaltrifluoromethanesulfonyl chloride (15.0 g) was added slowly maintaininga temperature below 5° C. The reaction mixture was stirred at roomtemperature for an additional hour. Diluted brine (1.0 L) was added andthe reaction mixture was stirred for 10 min at room temperature. Thelayers were separated; the organic layer was washed with diluted NaHCO₃(1.0 L) and then concentrated to ˜350 mL by rotary evaporation at 28° C.and stirred at room temperature for 30 min. Heptane (700 mL) was addedand the resulting slurry was stirred at room temperature for 30 min,cooled to 4° C. and stirred for 1 h. The solids were filtered, washedwith heptane, and then dried under vacuum to yield the title compound(193.0 g, >97% purity). The filtrate was concentrated, slurried in anisopropyl acetate and heptane mixture (1:3, 60 mL) over 30 min, filteredand dried to yield additional product (45.0 g, >97% purity).

c.trans-(7-Cyano-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester

Trifluoro-methanesulfonic acid7-tert-butoxycarbonylamino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ylester (236.6 g, 0.49 mol) was dissolved in N,N-dimethylformamide (851mL, 10.99 mol) and water (23.8 mL, 1.32 mol) at room temperature. Thesolution was purged with nitrogen for 5 min, and then connected to housevacuum for 5 min. Nitrogen purging and exposure to vacuum was repeatedtwice. To the reaction mixture was added zinc cyanide (34.2 g, 0.29mol), tris(dibenzylideneacetone)dipalladium(0) (4.4 g, 4.8 mmol) and1,1′-bis(diphenylphosphino)ferrocene (5.4 g, 9.7 mmol) with stirring.The reaction mixture was purged with nitrogen for 5 min, heated undernitrogen at 110° C. for 1 h, cooled to room temperature and thenfiltered through celite. The filtered reaction mixture was added slowlyto water (3 L), cooled to 0° C. with stirring, stirred for 30 min at 0°C., and then filtered. The filter cake was washed with water (500 mL)and dried in air for 2 h, slurried in ethanol (1 L) with stirring over 1h, and then filtered to give the title compound (165.0 g, >96% purity).The filtrate was dried (21.6 g) and dissolved in ethanol (110 mL) withstirring over 1 h, and the resulting slurry was filtered and dried undervacuum to give additional product (10.2 g, >98% purity).

Preparation 3:trans-(7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester

A slurry of the product of Preparation 2 (160.0 g, 446.3 mmol) andmethanol (3.3 L) was heated at 55° C. for 15 min, sodium perboratemonohydrate (280 g, 2800 mmol) and water (330 mL) was added and thereaction mixture was heated at 55° C. overnight. Additional sodiumperborate monohydrate (90 g) was added and the reaction mixture washeated at 55° C. overnight, then cooled to room temperature, and theinorganic solids were filtered off. The filtrate was transferred to a 5L flask and most of the solvent was removed by rotary evaporation. Tothe resulting slurry was added water (1.1 L) and ethyl acetate (450 mL)and the reaction mixture was stirred at room temperature for 20 min. Thereaction mixture was filtered and the filter cake was washed with water(200 mL) and then ethyl acetate (200 mL) and dried to yield the titlecompound (123 g, ˜95% purity). The filtrate was concentrated to drynessand dried under vacuum to yield additional product (18 g, 65% purity).

Preparation 4:trans-(7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester

To a mixture oftrans-(7-cyano-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester (33.0 g, 92 mmol), ethanol (45 mL), DMF (25 mL)and water (7.5 mL) was added hydrido(dimethylphosphoniousacid-kP)[hydrogen bis(dimethylphosphinito-kP)]platinum(II) (0.25 g, 0.58 mmol)and the reaction mixture was heated at 80° C. for 24 h. The reaction wascooled to room temperature and concentrated to dryness under vacuum togive the title compound (36.3 g) which was used without furtherpurification. (m/z): [M+H]⁺ calcd for C₂₁H₃₂N₂O₄ 377.24; found 377.8. ¹HNMR (d₆-DMSO, 400 mHz) δ (ppm): 7.92 (s, 1H), 7.64 (m, 2H), 7.26 (s,1H), 7.14 (d, J=7.9 Hz, 1H), 6.64 (d, J=9.4 Hz) 3.81 (t, J=10.0 Hz),3.58 (m, 1H), 3.30 (s, 3H), 2.58 (dd, J=16.9 Hz, 9.4 Hz, 1H), 1.82 (m,1H), 1.56-1.45 (m, 4H), 1.41 (s, 9H), 0.58 (m, 6H).

Preparation 5:trans-(7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester

To a solution oftrans-(7-cyano-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester (8.5 g, 24 mmol) in DMSO (105 mL) was added K₂CO₃(4.98 g, 36 mmol), and the mixture was stirred until all solids weredissolved. To the solution was added 30% hydrogen peroxide (12.2 mL, 120mmol) in 0.5 mL portions over 45 min at a rate to keep the temperature30-35° C. The reaction mixture was diluted with water (200 mL) andisopropyl acetate (500 mL), and sodium metabisulfite was added (10 g) toreduce excess peroxides. Layers were separated and the aqueous layer wasextracted with isopropyl acetate (3×150 mL) and 10% MeOH/isopropylacetate (2×100 mL). The combined organic layers were washed with water(3×150 mL) and saturated NaCl (100 mL), dried with Na₂SO₄ andconcentrated to give the title compound (9.4 g). (m/z): [M+H]⁺ calcd forC₂₁H₃₂N₂O₄ 377.24; found 377.6.

Preparation 6:trans-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide

Acetyl chloride (278.8 mL, 3920 mmol) was added dropwise to ethanol (382mL, 6530 mmol) at −5° C. over 2 h keeping the internal temperature below20° C. The resulting solution was added portion wise over 15 min,keeping the internal temperature below 30° C., to a slurry oftrans-(7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester (123.0 g, 327 mmol) and ethanol (500 mL) that hadbeen cooled to 10° C. The reaction mixture was stirred at roomtemperature for 2 h, and concentrated to ˜200 mL by rotary evaporation.Ethyl acetate (200 mL) was added and the resulting slurry was stirred at0° C. for 30 min, filtered and dried to yield the hydrochloride salt ofthe title compound (102 g, >98% purity) as a white solid.

Preparation 7: Carbonic acid 4-nitro-phenyl ester (R)-1-phenyl-ethylester

A mixture of (R)-1-phenyl-ethanol (60.6 g, 0.496 mol), pyridine (42.5mL, 0.526 mol) and 2-methyl-tetrahydrofuran (600 mL) was cooled to 0° C.and p-nitrophenyl chloroformate (100 g, 0.496 mol) was added over 15 minkeeping the internal temperature below 5° C. The reaction mixture waswarmed to room temperature and stirred for 2 h. To the reaction mixturewas added 1.0 M HCl in water (300 mL). Layers were separated. Theorganic layer was washed with 1N HCl (300 mL) and brine (300 mL),filtered, concentrated to dryness by rotary evaporation, and dried undervacuum to give the title compound (140 g) as a clear yellow oil.

Preparation 8:(6S,7S)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide a.((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester

A mixture of carbonic acid 4-nitro-phenyl ester (R)-1-phenyl-ethyl ester(102 g, 357 mmol), N,N-dimethylformamide (200 mL) and triethylamine(32.7 mL, 235 mmol) was stirred at room temperature overnight. To thereaction mixture was addedtrans-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (100 g, 320 mmol), N,N-dimethylformamide (320mL) and triethylamine (98.0 mL, 703 mmol). The reaction mixture washeated at 85° C. for 5 h and then stirred at room temperature overnight.Approximately 90% of the DMF was removed by distillation at 70° C. andthe resulting thick oil was cooled to room temperature and thenpartitioned between ethyl acetate (1.5 L) and diluted brine (500 mL).The organic layer was washed with 1M NaOH (3×500 mL) and dried withNa₂SO₄. Most of the solvent was removed by rotary evaporation, 3 volumesethyl acetate were added and resulting slurry was stirred at roomtemperature for 30 min, filtered and dried to give the title compound(48 g, >99% chemical and optical purity).

The filtrate was washed with 1M NaOH (200 mL) and then with dilutedbrine (2×200 mL). Most of the solvent was removed by rotary evaporationyielding a thick oil to which ethyl acetate (100 mL) was added. A pinchof seeds of the title compound was added and the reaction mixture wasrefrigerated at 0° C. after stirring for ˜30 min. The resulting thinslurry was stirred for 5 min and filtered; flask and filter cake werewashed with ethyl acetate (2×15 mL) to yield additional title compound(4.1 g, 97% chemical and >99% optical purity, 38% combined yield).

b.(6S,7S)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide

Acetyl chloride (193 mL, 2710 mmol) was added dropwise to ethanol (260mL, 4500 mmol) at −5° C. over 40 min keeping the internal temperaturebelow 30° C. The resulting solution was added over 5 min, at 10° C., toa mixture of((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester (49.0 g, 115 mmol) and ethanol (200 mL).The reaction mixture was stirred at room temperature overnight, andconcentrated to ˜100 mL by rotary evaporation. Ethyl acetate (100 mL)was added and the resulting slurry was stirred at 0° C. for 30 min andfiltered. The filter cake was washed with ethyl acetate and dried toyield the hydrochloride salt of the title compound (30 g, >99% purity).The volume of the filtrate was reduced almost to dryness. Isopropylalcohol (20 mL) was added and the resulting thick slurry was stirred for30 min and filtered. The filter cake was washed with ethyl acetate (2×20mL) and dried under vacuum overnight to yield additional title compound(5.5 g, >97% purity). ¹H NMR (DMSO-d₆): δ (ppm) 0.49 (t, 3H), 0.63 (t,3H), 1.62 (q, 2H), 1.89 (m, 1H), 2.09 (m, 1H), 2.60 (dd, 1H), 3.22 (m,1H), 3.41 (s, 3H), 3.50 (dd, 1H), 3.82 (q, 1H), 7.19 (d, 1H), 7.31 (br,1H), 7.70 (d, 1H), 7.71 (s, 1H), 7.98 (br, 1H), 8.15 (br, 3H).

Preparation 9:trans-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ol

To a solution oftrans-(1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester (6.0 g, 17.2 mmol) in dichloromethane (60 mL) wasadded a solution of 4.0 N HCl in dioxane (21.5 mL, 86 mmol) overapproximately 2 min. After stirring at room temperature overnight, thereaction mixture was concentrated at reduced pressure and dried undervacuum to give the hydrochloride salt of the title compound (5.5 g)(m/z): [M+H]⁺ calcd for C₁₅H₂₃NO₂ 250.36; found 250.2. ¹H NMR (d₆-DMSO,400 mHz) δ (ppm) 9.26 (s, 1H), 8.09 (br s, 3H), 6.92 (d, J=8.0 Hz, 1H),6.61 (m, 2H), 3.77 (m, 1H), 3.41 (s, 3H), 3.30 (dd, J=15.8 Hz, 5.9 Hz,1H), 3.17 (m, 1H), 2.43 (dd, J=15.5 Hz, 9.6 Hz, 1H), 1.85 (m, 2H),1.66-1.50 (m, 2H), 0.66 (t, J=7.4 Hz, 3H), 0.54 (t, J=7.1 Hz, 3H).

Preparation 10:(6S,7S)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-oland(6R,7R)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ola.((2R,3R)-1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester (RR) and((2S,3S)-1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester (SS)

A mixture oftrans-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-olhydrochloride salt (1.00 g, 3.5 mmol), carbonic acid 4-nitro-phenylester (R)-1-phenyl-ethyl ester (800 mg, 2.8 mmol), triethylamine (707mg, 7.0 mmol) and DMF (3.5 mL) was heated at 90° C. After 4 h, anadditional portion of carbonic acid 4-nitro-phenyl ester(R)-1-phenyl-ethyl ester (200 mg, 0.7 mmol) was added, and heatingcontinued for another 3 h. The reaction mixture was cooled and allowedto stand at room temperature overnight. The DMF was removed at reducedpressure and the residue was dissolved in ethyl acetate (25 mL). Theorganics were washed with 10% sodium carbonate and saturated sodiumchloride, dried with Na₂SO₄ and concentrated to dryness. The residue wasdissolved in methanol (6 mL) and a 1.0 N solution of sodium hydroxide inmethanol (3.0 mL, 3.0 mmol) was added. The reaction mixture was stirredat room temperature for 30 min, at which time 50% aqueous acetic acid (2mL) was added. The reaction mixture was concentrated to approximately 4mL, and 50% aqueous acetonitrile (15 mL) was added.

The crude diastereomers were separated by preparative HPLC and collectedseparately. Crude product was dissolved in 1:1 acetonitrile/water andseparated under the following conditions: column: Microsorb C18 100A 8μm column; flow rate: 50 mL/min; Solvent A: >99% water, 0.05% TFA;Solvent B: >99% acetonitrile, 0.05% TFA; Gradient (time(min)/% B): 0/15,4/15, 8/40, 60/55. The pure fractions of each were pooled and theacetonitrile was removed at reduced pressure. The product was extractedinto dichloromethane (3×30 mL), the organic extracts were dried withNa₂SO₄ and concentrated to give the title compounds.

RR: 435 mg (39% yield) (m/z): [M+H]⁺ calcd for C₂₄H₃₁NO₄ 398.52; found398.2. ¹H NMR (d₆-DMSO, 400 mHz) δ (ppm) 9.01 (s, 1H), 7.37-7.26 (m,5H), 7.05 (d, J=9.8 Hz, 1H), 6.86 (d, 8.2, 1H), 6.52 (dd, J=8.0, 2.4 Hz,1H), 6.48 (d, J=2.3 Hz, 1H), 5.70 (guar, J=6.7 Hz, 1H), 3.77 (t, J=10.3Hz, 1H), 3.55 (m, 1H), 3.32 (s, 3H), 3.17 (dd, J=15.9, 6.0 Hz, 1H), 2.43(m, 1H), 1.57-1.52 (m, 2H), 1.56 (d, J=6.7 Hz, 3H), 1.44-1.33 (m, 2H),0.60 (t, J=7.4 Hz, 3H), 0.51 (t, J=7.0 Hz, 3H).

SS: 363 mg (32% yield) (m/z): [M+H]⁺ calcd for C₂₄H₃₁NO₄ 398.52; found398.2. ¹H NMR (d₆-DMSO, 400 mHz) δ (ppm) 9.02 (s, 1H), 7.39-7.24 (m,5H), 7.03 (d, J=9.7 Hz, 1H), 6.85 (d, 8.3, 1H), 6.53 (dd, J=8.1, 2.6 Hz,1H), 6.48 (d, J=2.2 Hz, 1H), 5.69 (guar, J=6.7 Hz, 1H), 3.75 (t, J=10.6Hz, 1H), 3.52 (m, 1H), 3.27 (s, 3H), 3.14 (dd, J=15.9, 5.9 Hz, 1H), 2.37(dd, J=15.7, 9.5, 1H), 1.65-1.41 (m, 4H), 1.46 (d, J=6.6 Hz, 3H),0.64-0.60 (m, 6H).

b.(6S,7S)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ol

((2S,3S)-1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester (635 mg, 1.60 mmol) was treated with 4.0 NHCl in dioxane (6.0 mL, 24 mmol) and stirred at RT. After 3 days, thesolvent was removed at reduced pressure and the residual solid wastriturated with 50% dichloromethane in heptane (4 mL). The solid wascollected on a Buchner funnel and dried under vacuum to give thehydrochloride salt of the title compound (462 mg). (m/z): [M+H]⁺ calcdfor C₁₅H₂₃NO₂ 250.36; found 250.2. ¹H NMR (d₆-DMSO, 400 mHz) δ (ppm)9.23 (s, 1H), 8.02 (br s, 3H), 6.92 (d, J=8.2 Hz, 1H), 6.61 (m, 2H),3.77 (m, 1H), 3.41 (s, 3H), 3.30 (m, 1H), 3.17 (m, 1H), 2.44 (dd, J=15.9Hz, 9.8 Hz, 1H), 1.85 (m, 2H), 1.62-1.52 (m, 2H), 0.66 (t, J=7.2 Hz,3H), 0.55 (t, J=7.0 Hz, 3H).

c.(6R,7R)-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ol

Following the procedure of the previous step using((2R,3R)-1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester, the hydrochloride salt of the titlecompound was prepared. (m/z): [M+H]⁺ calcd for C₁₅H₂₃NO₂ 250.36; found250.4. ¹H NMR (d₆-DMSO, 400 mHz) δ (ppm) 9.23 (s, 1H), 8.02 (br s, 3H),6.92 (d, J=8.2 Hz, 1H), 6.61 (m, 2H), 3.77 (m, 1H), 3.41 (s, 3H), 3.30(m, 1H), 3.17 (m, 1H), 2.44 (dd, J=15.7 Hz, 10.2 Hz, 1H), 1.84 (m, 2H),1.62-1.52 (m, 2H), 0.66 (t, J=7.4 Hz, 3H), 0.55 (t, J=7.0 Hz, 3H).

Preparation 11:((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester (SS) and((2R,3R)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid (R)-1-phenyl-ethyl ester (RR)

A mixture of carbonic acid 4-nitro-phenyl ester (R)-1-phenyl-ethyl ester(7.35 g, 25.6 mol),trans-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (4.0 g, 13 mmol) and triethylamine (5.3 mL, 38mol) in DMF (13 mL) was heated at 85° C. After 2.5 hours, the reactionmixture was cooled and stirred at room temperature overnight. Solventwas removed under vacuum and the residue was purified by silica gelchromatography eluting with EtOAc in DCM (10% to 50% gradient) to give amixture containing the title compounds (6.96 g). The mixture ofdiastereomers was separated by preparative HPLC under the conditionsdescribed in Preparation 10 (a) except for use of the following gradient(time(min)/% B): 0/5, 4/5, 8/37, 60/42. The pure fractions of eachisomer were pooled and lyophylized to give the title compounds.

SS: 1.4 g (26%) (m/z): [M+H]⁺ calcd for C₂₅H₃₂N₂O₄ 425.24; found 425.6.

RR: 1.5 g (28%) (m/z): [M+H]⁺ calcd for C₂₅H₃₂N₂O₄ 425.24; found 425.4.

Single Crystal X-Ray Diffraction Analysis of Diastereomer SS

SS (3 mg) was dissolved in acetonitrile (100 mL) in an open HPLC vial,which was partially immersed in a 20 mL vial containing 1:9acetonitrile:water (4 mL). The 20 mL vial was capped and held at roomtemperature to provide large birefringent needle shaped crystals of SS.

X-ray diffraction crystal structure data was obtained for a singlecrystal with dimensions 0.44×0.13×0.10 mm using Mo K_(α) radiation(λ=0.71073 Å) on a Nonius KappaCCD diffractometer equipped with agraphite crystal and incident beam monochromator, and analyzed on aLINUX PC using SHELX97 software. The following lattice parameters werederived: unit cell is hexagonal with dimensions a=17.451 Å, b=17.451 Å,c=19.822 Å, α=90.00°, β=90.00°, γ=120.00°, cell volume (V)=5228 Å³,space group is P 3₁21. The molecule contains three chiral centers. Fromthe known R configuration of the carbon bearing the phenyl group:

the remaining two centers were determined to be in the S configuration.

Remaining crystals were analyzed by powder x-ray diffraction. Powderx-ray diffraction peaks predicted from the derived single crystalcrystallographic data were in good agreement with observed powder x-raydiffraction peaks.

Preparation 12:trans-7-Amino-8,8-dimethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide a. 7-Methoxy-1,1-dimethyl-3,4-dihydro-1H-napthalen-2-one

A slurry of sodium tert butoxide (21.1 g, 220 mmol) in THF (100 mL) wascooled to 0° C. A solution of 7-methoxy-3,4-dihydro-1H-napthalen-2-one(17.6 g, 100 mmol) and methyl iodide (30.1 g, 220 mmol) in THF (100 mL)was added dropwise over 40 min, and the reaction mixture was warmed toroom temperature after 10 min. Water (200 mL) and EtOAc (600 mL) wasadded. The layers were separated, the organic layer was washed withwater (5×100 mL) and saturated NaCl (100 mL), filtered and dried withNa₂SO₄ to provide the title compound (20 g).

b. 7-Methoxy-1,1-dimethyl-3,4-dihydro-1H-napthalen-2-one oxime

To a solution of 7-methoxy-1,1-dimethyl-3,4-dihydro-1H-napthalen-2-one(25.4 g, 98 mmol) in methanol (175 mL) was added a solution ofhydroxylamine hydrochloride (20.5 g, 295 mmol) and sodium acetate (24.2g, 295 mmol) in water (175 mL) and the reaction mixture was heated at70° C. for 3 h, and cooled in ice over 30 min. The solid was collectedon a Buchner funnel, stirred with methanol (125 mL) at 50° C. for 30min, and then stirred at RT overnight. The reaction mixture was cooledto 0° C.; solid was collected on a Buchner funnel, rinsed with coldmethanol (20 mL) and dried under vacuum to give the title compound (14.7g).

c.(1aS,7aR)-4-Methoxy-2,2-dimethyl-1a,2,7,7a-tetrahydro-1H-1-aza-cyclopropa[b]-naphthalene

To a solution of 7-methoxy-1,1-dimethyl-3,4-dihydro-1H-napthalen-2-oneoxime (15.3 g, 70 mmol) in THF (240 mL) was added diethylamine (18 mL).The reaction mixture was cooled to 0° C. and a 2.0 M solution of lithiumaluminum hydride in THF (100 mL, 200 mmol) was added slowly over 20 minto control the rate of hydrogen evolution. The reaction mixture washeated to 70° C. for 1 h, cooled to 0° C. and Na₂SO₄ 10H₂O (20 g), brine(60 mL), and EtOAc (300 mL) were added. The solid were washed with EtOAc(4×100 mL); the combined organic layers were washed with water (4×100mL) and brine (100 mL), dried with Na₂SO₄, and concentrated to givecrude title product (14.3 g). The crude product was dissolved in EtOAc(500 mL), extracted with 0.1 N HCl (100 mL), then with 0.3 N HCl (225mL). Sodium carbonate (8 g, 75 mmol) was added to the aqueous layerwhich was extracted with EtOAc (4×200 mL). Organic layers were combined,dried with Na₂SO₄ and concentrated to give the title compound as an oil(10.1 g) which crystallized on standing to a tan solid. (m/z): [M+H]⁺calcd for C₁₃H₁₇NO 204.14; found 204.2

d.trans-(7-Hydroxy-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester

Using a procedure similar to that of Preparations 1(b) and 2(a), thetitle compound was prepared. ¹H NMR (d₆-DMSO, 400 mHz) δ (ppm): 9.04 (s,1H), 6.83 (d, J=8.2 Hz, 1H), 6.69 (d, J=9.4 Hz, 1H), 6.65 (d, J=2.5 Hz,1H), 6.52 (dd, J=8.2, 2.5 Hz, 1H), 3.50 (m, 1H), 3.45 (m, 1H), 3.30 (s,3H), 3.15 (m, 1H), 2.55 (m, 1H), 1.34 (s, 9H), 1.16 (s, 3H), 1.00 (s,3H).

c.trans-(7-Carbamoyl-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester

Using a procedure similar to that of Preparations 2(b), 2(c), and 5, thetitle compound was prepared. (m/z): [M+H]⁺ calcd for C₁₉H₂₈N₂O₄ 349.21;found 349.1.

f.trans-(7-Amino-6-methoxy-8,8-dimethyl-5,6,7,8-tetrahydronaphthalene-2-carboxylicacid amide

To a slurry oftrans-(7-carbamoyl-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamicacid tert-butyl ester (9.22 g, 26.4 mmol) in DCM (100 mL) was added 4 NHCl in dioxane (25 mL, 100 mmol) slowly. The reaction mixture wasstirred at RT for 15 h, concentrated to dryness, triturated with DCM (25mL) for 30 min, filtered, rinsed with DCM (3×15 mL), and dried undervacuum. Ethanol (100 mL) was added and the reaction mixture wasconcentrated under vacuum to give the HCl salt of the title compound(7.17 g) as a white powder. (m/z): [M+H]⁺ calcd for C₁₄H₂₀N₂O₂ 249.16;found 249.1. ¹H NMR (d₆-DMSO, 400 mHz) δ (ppm): 8.18 (s, 3H), 8.00 (s,1H), 7.92 (d, J=1.6 Hz, 1H), 7.66 (dd, J=8.0 Hz, 1.8 Hz, 1H), 7.32 (s,1H), 7.17 (d, J=8.0 Hz, 1H), 3.70 (m, 1H), 3.44 (s, 3H), 3.43 (m, 1H),3.22 (m, 1H), 2.67 (dd, J=16.4 Hz, 10.2 Hz), 1.50 (s, 3H), 1.24 (s, 3H).

Example 1trans-6-Methoxy-8,8-dimethyl-7-(3-oxo-3-piperidin-1-yl-propylamino)-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide a.3-(trans-7-Carbamoyl-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl ester

A mixture oftrans-7-amino-6-methoxy-8,8-dimethyl-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (0.25 g, 0.88 mmol) anhydrous ethanol (0.90mL), triethylamine (0.14 mL, 1.32 mmol) and acrylic acid ethyl ester(0.14 mL, 1.0 mmol) were heated at 70° C. After 23 h, additional acrylicacid ethyl ester (0.04 mL, 0.3 mmol) was added and heating wascontinued. Five hours after the addition, the reaction was cooled toroom temperature, combined with another run (0.36 mmol scale) andconcentrated under vacuum. The title compound (0.61 g) was used withoutfurther purification. (m/z): [M+H]⁺ calcd for C₁₉H₂₈N₂O₄ 349.21; found349.0.

b.3-(trans-7-Carbamoyl-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid

To a slurry of3-(trans-7-carbamoyl-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl ester (0.60 g, 1.24 mmol) in methanol (3 mL) and water (1 mL)was added 2N NaOH (2 mL, 4 mmol). After 1 h, the reaction mixture wasconcentrated to −3 mL, dissolved in 50% aqueous AcOH and purified bypreparative HPLC to give the title compound as the TFA salt (0.40 g,74%). (m/z): [M+H]⁺ calcd for C₁₇H₂₄N₂O₄ 321.18; found 321.0. ¹H NMR(d₆-DMSO, 400 mHz) δ (ppm): 9.05 (br s, 1H), 7.94 (s, 1H), 7.89 (d,J=1.6 Hz, 1H) 7.65 (br s, 1H), 7.64 (dd, J=7.8 Hz, 1.6 Hz, 1H), 7.31 (s,1H), 7.16 (d, J=8.0 Hz, 1H), 3.83 (m, 1H), 3.57-3.32 (m, 4H), 3.40 (s,3H), 2.79-2.50 (m, 3H), 1.49 (s, 3H), 1.24 (s, 3H).

c.trans-6-Methoxy-8,8-dimethyl-7-(3-oxo-3-piperidin-1-yl-propylamino)-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide

To a solution of racemic3-(trans-7-carbamoyl-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid TFA salt (40 mg, 0.092 mmol) in DMF (1 mL) was addedN,N-diisopropylethylamine (35 μL, 0.020 mmol), piperidine (20 μL, 0.020mmol) and a 0.5 N solution ofN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uraniumhexafluorophosphate (HATU) (0.22 mL, 0.11 mmol) and the reaction mixturewas allowed to stand at room temperature for 15 hours. The reaction wasdiluted with 50% aqueous AcOH and purified by preparative HPLC to givethe title compound as the TFA salt (45 mg, 97%). (m/z): [M+H]⁺ calcd forC₂₂H₃₃N₃O₃ 388.26; found 388.5.

Examples 2-9

Following the procedure of Example 1, the compounds of Examples 2-9 wereprepared.

TABLE 1

Ex Calc Obs No R⁸ R⁹ Formula [M + H]⁺ [M + H]⁺ 2 2,2- H C₂₂H₃₅N₃O₃390.28 390.3 dimethylpropyl 3 benzyl H C₂₄H₃₁N₃O₃ 410.24 410.4 4 benzylmethyl C₂₅H₃₃N₃O₃ 424.25 424.2 5 ethyl ethyl C₂₁H₃₃N₃O₃ 376.26 376.4 6n-propyl n-propyl C₂₃H₃₇N₃O₃ 404.29 404.8 7 n-butyl H C₂₁H₃₃N₃O₃ 376.26376.4 8 n-butyl methyl C₂₂H₃₅N₃O₃ 390.28 391.0 9 n-hexyl H C₂₃H₃₇N₃O₃404.29 404.8

Example 102-Benzyl-3-(trans-1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl ester a. Preparation of Ethyl (Phenylmethyl)Propanedioic Acid

To a solution of diethyl benzylmalonate (20.0 g, 80 mmol) in ethanol(500 mL) was added potassium hydroxide (4.7 g, 84 mmol) as pellets. Themixture was stirred at room temperature for 24 h, and concentrated todryness in vacuo. The residue was dissolved in water (300 mL),transferred to a separatory funnel, and washed with ether (2×150 mL).The aqueous solution was acidified to pH˜2 by adding conc. HCl, andextracted with ether (2×400 mL). The organic layer was dried over MgSO₄,filtered and evaporated to dryness in vacuo, yielding the title compoundas an oil residue (16.9 g, 95%) which was used without furtherpurification.

b. Preparation of 2-benzyl-acrylic acid ethyl ester

The product of the previous step (10 g, 45 mmol) in a round bottomedflask was cooled in an ice bath and then diethylamine (4.8 mL) and 37%aqueous formaldehyde solution (4.8 mL) was added over 10 min withstirring. After stirring for 7 h, the mixture was diluted with water(100 mL), and extracted with ether (500 mL). The organic layer waswashed with 2 M HCl (300 mL), saturated sodium bicarbonate (300 mL), andbrine solution, dried over MgSO₄, and evaporated to dryness, to providethe title compound as an oil (6.07 g, 71%). ¹H NMR (CD₃OD) δ (ppm)7.15-7.08 (m, 3H), 7.07-7.05 (m, 2H), 6.07 (d, J=1.5 Hz, 1H), 5.41 (d,J=1.5 Hz, 1H), 4.06-3.99 (q, J=6.6 Hz, 2H), 3.50 (s, 2H), 1.14-1.07 (t,J=6.6 Hz, 2H).

c.2-Benzyl-3-(trans-1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl ester

A mixture oftrans-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-olhydrochloride (0.12 g, 0.42 mmol), 2-benzyl-acrylic acid ethyl ester(0.40 g, 2.1 mmol), and N,N-disopropylethylamine (0.17 mL, 1.0 mmol) inethanol (2 mL) was heated at 100° C. for 4 days, cooled to roomtemperature and diluted with 1:1 H₂O/ACN. The crude solution waspurified by preparative HPLC to give the title compound as the TFA salt(35 mg, 15% yield).

Example 112-Benzyl-3-(trans-1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid

A mixture of2-benzyl-3-(trans-1,1-diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl esterTFA (0.035 g, 0.063 mmol) and 3 N sodium hydroxide (0.21mL) in tetrahydrofuran (0.21 mL) was heated at 40° C. for 4 h, andcooled to room temperature. The tetrahydrofuran was removed underreduced pressure and the pH of the remaining aqueous layer was adjustedto 8 with 1 N sodium hydroxide. The product was extracted withacetonitrile (2×0.5 mL) and concentrated to dryness to give the titlecompound (20.0 mg, 77.2% yield). (m/z): [M+H]⁺ calcd for C₂₅H₃₃NO₄,412.25; found, 412.5.

Example 122-Benzyl-3-(trans-1,1-dimethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl ester

Following the procedure of Example 10 usingtrans-7-amino-8,8-dimethyl-6-methoxy-5,6,7,8-tetrahydronaphthalene-2-carboxylicacid amide hydrochloride the TFA salt of the title compound wasprepared. (m/z): [M+H]⁺ calcd for C₂₅H₃₃NO₄, 412.25; found, 412.5.

Example 132-Benzyl-3-(trans-1,1-dimethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl ester

Following the procedure of Example 11 using2-benzyl-3-(trans-1,1-dimethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-propionicacid ethyl ester TFA, the TFA salt of the title compound was prepared.(m/z): [M+H]¹ calcd for C₂₃H₂₉NO₄, 384.22; found, 384.5.

Example 141-[2-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]-cyclohexyl-aceticacid a. (1-Benzyloxycarbonylmethyl-cyclohexyl)-acetic acid

A mixture of 1,1-cyclohexanediacetic anhydride (5.0 g, 27 mmol),pyridine (2.2 mL, 27 mmol), and benzyl alcohol (2.8 mL, 27 mmol) andtoluene (5.0 mL, 47 mmol) was stirred at 80° C. overnight. The reactionmixture was cooled to room temperature and partitioned between ethylacetate and 1 N HCl. The organic layer was washed three times with 1 NHCl and brine. The organic extract was dried over sodium sulfate,filtered, and concentrated to dryness to yield the title compound (7.06g, 89% yield) as a clear oil. ¹H NMR (DMSO-d₆, 400 mHz) δ (ppm): 11.96(s, 1H), 7.32 (m, 5H), 5.03 (s, 2H), 2.52 (s, 2H), 2.34 (s, 2H), 1.40(m, 10H).

b. [1-(2-Hydroxy-ethyl)-cyclohexyl]-acetic acid benzyl ester

A solution of (1-benzyloxycarbonylmethyl-cyclohexyl)-acetic acid (3.0 g,10 mmol) in tetrahydrofuran (20 mL) was cooled to 0° C. and 1 Mborane-THF complex in tetrahydrofuran (21 mL, 20 mmol) was addeddropwise over 5 min. The reaction mixture was allowed to warm to roomtemperature, stirred for 1 h at room temperature. and cooled to 0° C.Methanol was added slowly to quench the reaction. The reaction mixturewas warmed to room temperature, stirred for 30 min, and concentrated todryness to provide the title compound (2.2 g).

c. [1-(2-Oxo-ethyl)-cyclohexyl]-acetic acid benzyl ester

A mixture of [1-(2-hydroxy-ethyl)-cyclohexyl]-acetic acid benzyl ester(1.3 g, 47 mmol), dimethyl sulfoxide (0.83 mL, 12 mmol), andN,N-diisopropylethylamine (2.0 mL, 12 mmol) in dichloromethane (20 mL)was cooled to 0° C. and sulfur trioxide-pyridine complex (1.9 g, 12mmol) was added. The reaction mixture was stirred at 0° C. for 2 h andthe reaction was quenched with 0.1 N HCl. The aqueous layer wasdiscarded. The organic extract was washed twice with 0.1 N HCl andbrine. The organic extract was dried with sodium sulfate, filtered, andconcentrated to dryness to provide the title compound (1.17 g).

d.1-[2-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]-cyclohexyl-aceticacid

To a mixture of [1-(2-oxo-ethyl)-cyclohexyl]-acetic acid benzyl ester(0.088 g, 0.32 mmol),(6S,7S)-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (0.050 g; 0.16 mmol), and triethylamine (0.022mL, 0.16 mmol) in dichloromethane (1.2 mL) and methanol (0.5 mL) wasadded sodium triacetoxyborohydride (0.083 g, 0.39 mmol). The reactionmixture was stirred overnight at room temperature. Saturated sodiumcarbonate (10 mL) and dichloromethane (20 mL) were added. The organicextract was collected. The aqueous layer was extracted with additionaldichloromethane (10 mL). The organic extracts were combined, dried withsodium sulfate, filtered and concentrated to dryness. The crude residuewas dissolved in ethanol. Lithium hydroxide (0.040 g, 2.0 mmol) wasadded. The reaction mixture was stirred overnight at room temperatureand concentrated to dryness. The residue was dissolved in 1:1 AcOH/H₂Oand purified by preparative HPLC to give the title compound as the TFAsalt (10 mg, 11% yield over 2 steps). (m/z): [M+H]⁺ calcd forC₂₆H₄₀N₂O₄, 445.30; found, 445.5.

Examples 15-16

Following the procedure of Example 14 using the appropriateaminotetralin hydrochloride, the TFA salts of the title compounds wereprepared.

Example 15

{1-[2-(trans-1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]-cyclohexyl}-aceticacid (m/z): [M+H]⁺ calcd for C₂₅H₃₉NO₄, 418.29; found, 418.3.

Example 16

{1-[2-(1,1-Dimethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]cyclohexyl}-aceticacid (m/z): [M+H]⁺ calcd for C₂₃H₃₅NO₄, 390.26; found, 390.6.

Example 17N-Cyclohexyl-2-{1-[2-((2S,3S)-7-hydroxy-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]-cyclohexyl}-acetamidea. (1-Cyclohexylcarbamoylmethyl-cyclohexyl)-acetic acid

A mixture of 1,1-cyclohexanediacetic anhydride (0.911 g, 5.0 mmol) andcyclohexylamine (0.63 mL, 5.5 mmol) in dichloromethane (10.0 mL) wasstirred overnight at room temperature. The reaction mixture was dilutedwith H₂O (5 mL) and the aqueous layer was acidified to pH-5 using 6 NHCl. The organic layer was collected. The aqueous layer was extractedwith additional dichloromethane (5 mL). The organic extracts werecombined, dried with sodium sulfate, filtered and concentrated todryness to give crude title compound (1.63 g, 95% purity). (m/z): [M+H]⁺calcd for C₁₆H₂₇NO₃, 282.21; found 282.5.

b. N-Cyclohexyl-2-[1-(2-oxo-ethyl)-cyclohexyl]-acetamide

Following the procedure of Example 14 (b) and (c), the title compoundwas prepared. (m/z): [M+H]⁺ calcd for C₁₆H₂₇NO₂, 266.20; found 266.3

c.N-Cyclohexyl-2-{1-[2-(trans-7-hydroxy-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]-cyclohexyl}-acetamide

To a mixture of N-cyclohexyl-2-[1-(2-oxo-ethyl)-cyclohexyl]-acetamide(0.09 g, 0.33 mmol),trans-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-ol.hydrochloride(0.078 g; 0.30 mmol), and N,N-diisopropylamine (0.11 mL, 0.61 mmol) indichloromethane (2 mL) at 0° C., sodium triacetoxyborohydride (0.32 g,1.51 mmol) was added. The reaction mixture was allowed to warm to roomtemperature and stirred for 45 min. Water and 0.1 N sodium hydroxide wasadded to quench the reaction, keeping the pH<9. The suspension wasdiluted with dichloromethane (10 mL). The organic extract was collected.The aqueous layer was extracted with additional dichloromethane (10 mL).The organic extracts were combined, dried with sodium sulfate, filteredand concentrated to dryness. The residue was dissolved in 1:1 AcOH/H₂Oand purified by preparative HPLC to give the title compound as the TFAsalt (22 mg, 12% yield). (m/z): [M+H]⁺ calcd for C₂₉H₄₆N₂O₃, 471.35;found, 471.4.

Example 182-{1-[2-(7-Hydroxy-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]-cyclohexyl}-1-(4-methyl-piperazin-1-yl)-ethanone

Following the procedure of Examples 14 and 17 using 1-methylpiperazinein step (a), the title compound was prepared. (m/z): [M+H]⁺ calcd forC₂₈H₄₅N₃O₃, 472.35; found, 472.3.

Example 194-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2,2-diphenyl-butyricacid methyl ester (19-A) and4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2,2-diphenyl-butyricacid (19-B) a. 4-Bromo-2,2-diphenyl-butyric acid methyl ester

4-Bromo-2,2-diphenylbutyric acid (5.0 g, 16 mmol), thionyl chloride (1.1mL, 16 mmol), and N,N-dimethylformamide (0.02 mL, 0.20 mmol) inchloroform (16 mL) were mixed at room temperature under a stream ofnitrogen. The reaction mixture was heated to reflux, stirred underreflux for 4 h, and cooled to 0° C. Methanol (50 mL) was added. Thereaction mixture was warmed to room temperature and concentrated todryness, yielding a brown oil. The crude material was purified on SiO₂(120 g) using 10% EtOAc/Hexanes as eluent to provide the title compound(3.9 g) as a clear oil that crystallized on standing. ¹H NMR (DMSO-d₆,400 mHz) δ (ppm): 7.33-7.20 (m, 10H), 3.70 (s, 3H), 3.12-3.08 (m, 2H),2.98-2.93 (m, 2H).

b. 2,2-Diphenyl-4-oxo-butyric acid methyl ester

A mixture of 4-bromo-2,2-diphenyl-butyric acid methyl ester (0.10 g,0.300 mmol) and trimethylamine N-oxide (0.09 g, 1.2 mmol) in dimethylsulfoxide (0.5 mL, 7 mmol) was stirred overnight at room temperature.The reaction was quenched with cold brine. The crude product wasextracted three times with dichloromethane, dried over a bed of Na₂SO₄,filtered, and concentrated to dryness to provide crude title compoundwhich was used without further purification.

c.4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2,2-diphenyl-butyricacid methyl ester (19-A) and4-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-2,2-diphenyl-butyricacid. (19-B)

To a mixture of(6S,7S)-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (0.025 g, 0.08 mmol),2,2-diphenyl-4-oxo-butyric acid methyl ester (0.021 g, 0.08 mmol), andtriethylamine (0.011 mL, 0.08 mmol) in dichloromethane (1.0 mL) andmethanol (0.5 mL) at room temperature was added sodiumtriacetoxyborohydride (0.092 g, 0.44 mmol). The reaction mixture wasstirred overnight at room temperature. The reaction was quenched withsaturated sodium bicarbonate. The reaction mixture was diluted withdichloromethane. The aqueous layer was discarded. The organic extractwas washed twice with brine, dried over sodium sulfate, filtered, andconcentrated to dryness. The crude product was dissolved in methanol (2mL), and 5 N sodium hydroxide (0.10 mL) was added. The reaction mixturewas heated at 50° C. overnight, diluted with 1:1 AcOH/H₂O (1.5 mL),filtered, and purified by preparative HPLC to give the title compoundsas their TFA salts.

19-A: (m/z): [M+H]⁺ calcd for C₃₃H₄₀N₂O₄, 529.30; found, 529.1.

19-B: (m/z): [M+H]⁺ calcd for C₃₂H₃₈N₂O₄, 515.28; found, 515.2.

Example 20trans-7-(3-Dimethylcarbamoyl-3,3-diphenyl-propylamino)-6-methoxy-8,8-dimethyl-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide

A mixture oftrans-7-amino-8,8-dimethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (0.10 g; 0.39 mmol),(3,3-diphenyldihydrofuran-2-ylidine)dimethylammonium bromide (0.14 g,0.39 mmol) and sodium carbonate (0.082 g, 0.79 mmol) inN,N-dimethylformamide (1.0 mL) was heated at 80° C. and stirredovernight. The reaction mixture was cooled to room temperature,filtered, and diluted with 1:1 AcOH/H₂O (3 mL). The crude solution waspurified by preparative HPLC to give the title compound as the TFA salt(29 mg, 12% yield). (m/z): [M+H]⁺ calcd for C₃₂H₃₉N₃O₃, 514.30; found,514.4.

Example 214-(trans-7-Hydroxy-3-methoxy-1,1-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-N,N-dimethyl-2,2-diphenyl-butyramide

Following the procedure of Example 20 usingtrans-7-amino-8,8-dimethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-olhydrochloride the title compound was prepared. (m/z): [M+H]+ calcd forC₃₁H₃₈N₂O₃, 487.29; found, 487.3.

Example 22trans-(1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-heptanoicacid amide a.trans-7-(1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-heptanenitrile

A mixture oftrans-7-amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalen-2-01hydrochloride (0.15 g; 0.53 mmol), 7-bromoheptanenitrile (0.10 g, 0.53mmol) and sodium carbonate (0.11 g, 1.52 mmol) in N,N-dimethylformamide(2.0 mL) was heated at 80° C. and stirred overnight. The reactionmixture was cooled to room temperature, filtered, and diluted with 1:1AcOH/H₂O (3 mL). The crude solution was purified by preparative HPLC togive the title compound as the TFA salt (39 mg, 16% yield). (m/z):[M+H]⁺ calcd for C₂₂H₃₄N₂O₂, 359.26; found, 359.4.

b.trans-(1,1-Diethyl-7-hydroxy-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-heptanoicacid amide

To a solution of the product of the previous step (39 mg, 0.083 mmol) indimethyl sulfoxide (1.0 mL), potassium carbonate (23 mg, 0.16 mmol) wasadded and then hydrogen peroxide (30 wt. % solution in water, 0.10 mL,0.91 mmol) was added dropwise. The reaction mixture was stirredovernight at room temperature. The reaction was quenched with saturatedsodium sulfite solution. The reaction mixture was diluted with 1:1AcOH/H₂O (4.0 mL) and purified by preparative HPLC to give the titlecompound as the TFA salt (6.3 mg, 16% yield). (m/z): [M+H]¹ calcd forC₂₂H₃₆N₂O₃, 377.27; found, 377.5.

Example 237-(5-Carbamoyl-pentylamino)-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide

A mixture oftrans-7-Amino-8,8-diethyl-6-methoxy-5,6,7,8-tetrahydro-naphthalene-2-carboxylicacid amide hydrochloride (50 mg, 0.16 mmol), 6-bromohexanamide (31 mg,0.16 mmol) and sodium carbonate (34 mg, 0.32 mmol) in dimethylformamide(1.0 mL) was heated at 80° C. and stirred overnight. The reactionmixture was cooled to room temperature, filtered, and diluted with 1:1AcOH/H₂O (3 mL). The crude solution was purified by preparative HPLC togive the title compound as the TFA salt (11 mg, 14% yield). (m/z):[M+H]⁺ calcd for C₂₂H₃₅N₃O₃, 390.27; found, 390.4.

Examples 24-31

Following the procedures of Example 22 (Examples 24-28) or Example 23(Examples 29-31) the compounds of Table 2 were prepared.

TABLE 2

Ex Calc Obs No R¹ R², R³  p  Formula [M + H]⁺ [M + H]⁺ 24 —C(O)NH₂ Et,Et 4 C₂₃H₃₇N₃O₃ 404.28 404.4 25 —C(O)NH₂ Et, Et 2 C₂₁H₃₃N₃O₃ 376.25376.2 26 —OH Et, Et 2 C₂₀H₃₂N₂O₃ 349.24 349.3 27 —OH Et, Et 1 C₁₉H₃₀N₂O₃335.23 335.2 28 —C(O)NH₂ Et, Et 1 C₂₀H₃₁N₃O₃ 362.24 362.4 29 —OH Et, Et3 C₂₁H₃₄N₂O₃ 363.26 363.3 30 —C(O)NH₂ Me, Me 3 C₂₀H₃₁N₃O₃ 362.24 362.431 —OH Me, Me 3 C₁₉H₃₀N₂O₃ 335.23 335.5

Example 325-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-3-(4-chloro-phenyl)-pentanoicacid a. 3-(4-Chloro-phenyl)-pentanedioic acid monoethyl ester

A solution of 3-(4-chlorophenyl)glutaric acid (1.0 g, 4.1 mmol) inacetic anhydride (20 mL, 200 mmol) was heated to reflux, stirred for 17h, cooled at room temperature, and concentrated to dryness. The cruderesidue was dried under vacuum for 1 h. The crude anhydride wasdissolved in ethanol (30 mL) and triethylamine (3.0 mL, 22 mmol) wasadded. The reaction was stirred for 17 h at room temperature and thenconcentrated to dryness. The crude residue was dissolved indichloromethane (5 mL) and purified on SiO₂ (40 g) using 5% MeOH/DCM toprovide the title product (0.785 g, 70% yield) as a clear oil. ¹H NMR(DMSO-d₆, 400 mHz) δ (ppm): 12.09 (s, 1H), 7.30-7.22 (m, 4H), 3.93-3.87(m, 2H), 3.4 (m, 1H), 2.71-2.46 (s, 4H), 1.0 (t, J=7 Hz, 3H).

b. 3-(4-Chloro-phenyl)-5-oxo-pentanoic acid ethyl ester

Following the procedure of Example 14 (b) and (c), the title compoundwas prepared. ¹H NMR (DMSO-d₆, 400 mHz) δ (ppm): aldehyde proton at 9.47ppm.

c.5-((2S,3S)-7-Carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-3-(4-chloro-phenyl)-pentanoicacid

Following the procedure of Example 14 (d) using3-(4-chloro-phenyl)-5-oxo-pentanoic acid ethyl ester the title compoundwas prepared. (m/z): [M+1-1]⁺ calcd for C₂₇H₃₅ClN₂O₄, 487.23; found,487.3.

Examples 33 and 34

Following procedures similar to those used in the previous example, thefollowing compounds were prepared.

Example 33

5-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-3-phenyl-pentanoicacid (m/z): [M+H]⁺ calcd for C₂₇H₃₆N₂O₄, 453.28; found, 453.4.

Example 34

1-[2-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-ethyl]-cyclohexanecarboxylicacid (m/z): [M+H]⁺ calcd for C₂₅H₃₈N₂O₄, 431.29; found, 431.6.

Assay 1: Radioligand Binding Assay on Human Mu, Human Delta and GuineaPig Kappa Opioid Receptors

a. Membrane Preparation

CHO-K1 (Chinese Hamster Ovary) cells stably transfected with human muopioid or with guinea pig kappa receptor cDNA were grown in mediumconsisting of Ham's-F12 media supplemented with 10% FBS, 100 units/mlpenicillin-100 μg/mL streptomycin and 800 μg/mL Geneticin in a 5% CO₂,humidified incubator @ 37° C. Receptor expression levels (B_(max)˜2.0and ˜0.414 μmol/mg protein, respectively) were determined using[³H]-Diprenorphine (specific activity ˜50-55 Ci/mmol) in a membraneradioligand binding assay.

Cells were grown to 80-95% confluency (<25 subculture passages). Forcell line passaging, the cell monolayer was incubated for 5 minutes atroom temperature and harvested by mechanical agitation in 10 mL of PBSsupplemented with 5 mM EDTA. Following resuspension, cells weretransferred to 40 mL fresh growth media for centrifugation for 5 minutesat 1000 rpm and resuspended in fresh growth medium at the appropriatesplit ratio.

For membrane preparation, cells were harvested by gentle mechanicalagitation with 5 mM EDTA in PBS followed by centrifugation (2500 g for 5minutes). The pellets were resuspended in Assay Buffer (50 mM4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acidN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES)), pH7.4, and homogenized with a polytron disrupter on ice. The resultanthomogenates were centrifuged (1200 g for 5 minutes), the pelletsdiscarded and the supernatant centrifuged (40,000 g for 20 minutes). Thepellets were washed once by resuspension in Assay Buffer, followed by anadditional centrifugation (40,000 g for 20 minutes). The final pelletswere resuspended in Assay Buffer (equivalent 1 T-225 flask/1 mL assaybuffer). Protein concentration was determined using a Bio-Rad BradfordProtein Assay kit and membranes were stored in frozen aliquots at −80°C., until required.

Human delta opioid receptor (hDOP) membranes were purchased from PerkinElmer. The reported K_(d) and B_(max) for these membranes determined bysaturation analyses in a [³H]-Natrindole radioligand binding assays were0.14 nM (pK_(d)=9.85) and 2.2 pmol/mg protein, respectively. Proteinconcentration was determined using a Bio-Rad Bradford Protein Assay kit.Membranes were stored in frozen aliquots at −80° C., until required.

b. Radioligand Binding Assays

Radioligand binding assays were performed in an Axygen 1.1 mL deep well96-well polypropylene assay plate in a total assay volume of 200 μLcontaining the appropriate amount of membrane protein (˜3, ˜2 and ˜20 μgfor mu, delta and kappa, respectively) in Assay Buffer, supplementedwith 0.025% bovine serum albumin (BSA). Saturation binding studies fordetermination of K_(d) values of the radioligand were performed using[³H]-Diprenorphine at 8-12 different concentrations ranging from 0.001nM-5 nM. Displacement assays for determination of pKi values ofcompounds were performed with [³H]-Diprenorphine at 0.5, 1.2, and 0.7 nMfor mu, delta, and kappa, respectively, and eleven concentrations ofcompound ranging from 10 pM-100 μM.

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The curveminimum was fixed to the value for nonspecific binding, as determined inthe presence of 10 μM naloxone. K_(i) values for test compounds werecalculated, in Prism, from the best fit IC₅₀ values, and the K_(d) valueof the radioligand, using the Cheng-Prusoff equation(K_(i)=IC₅₀/(1+([L]/K_(d))) where [L]=the concentration of[³H]-Diprenorphine Results are expressed as the negative decadiclogarithm of the K_(i) values, pK_(i).

Test compounds having a higher pK_(i) value in these assays have ahigher binding affinity for the mu, delta, or kappa opioid receptor. Thefinal compounds named in Examples 1-31 were tested in these assays. Allof the compounds had a pK_(i) value between about 7.4 and about 10.5 atthe human mu opioid receptor. For example, the compounds of Examples 1,11, and 24 had pK_(i) values of 10.0, 8.9, and 9.1, respectively.Compounds of the invention also exhibited pK_(i) values between about6.3 and about 10.2 at the human delta and guinea pig kappa opioidreceptors.

Assay 2: Agonist Mediated Activation of the Mu-Opioid Receptor inMembranes Prepared from CHO-K1 Cells Expressing the Human Mu-OpioidReceptor

In this assay, the potency and intrinsic activity values of testcompounds were determined by measuring the amount of bound GTP-Eu orbound [³⁵S]GTPγS present following receptor activation in membranesprepared from CHO-K1 cells expressing the human mu opioid receptor. Adescription of the GTP-Eu assay follows. An analogous protocol wasfollowed using the radioligand [³⁵S]GTPγS in which the amount of bound[³⁵S]GTPγS was determined using a Packard Topcount liquid scintillationcounter.

a. Mu Opioid Receptor Membrane Preparation:

Human mu opioid receptor (hMOP) membranes were either prepared asdescribed above or were purchased from Perkin Elmer. The reported pK_(d)and B_(max) for the purchased membranes determined by saturationanalyses in a [³H]-Diprenorphine radioligand binding assays was 10.06and 2.4 μmol/mg protein, respectively. Protein concentration wasdetermined using a Bio-Rad Bradford Protein Assay kit. Membranes werestored in frozen aliquots at −80° C., until required. Lyophilized GTP-Euand GDP were diluted to 10 μM and 2 mM, respectively, in doubledistilled H₂O then mixed and permitted to sit at room temperature for 30minutes prior to transfer to individual aliquots samples for storage at−20° C.

b. Human Mu GTP-Eu Nucleotide Exchange Assay

GTP-Eu nucleotide exchange assays were performed using the DELPHIAGTP-binding kit (Perkin/Elmer) in AcroWell 96 well filter platesaccording to the manufacturer's specifications. Membranes were preparedas described above, and prior to the start of the assay, aliquots werediluted to a concentration of 200 μg/mL in Assay Buffer (50 mM HEPES, pH7.4 at 25° C.), then homogenized for 10 seconds using a Polytronhomogenizer. Test compounds were received as 10 mM stock solutions inDMSO, diluted to 400 μM into Assay Buffer containing 0.1% BSA, andserial (1:5) dilutions then made to generate ten concentrations ofcompound ranging from 40 pM⁻ 80 μM-GDP and GTP-Eu were diluted to 4 μMand 40 nM, respectively, in Assay Buffer. The assay was performed in atotal volume of 100 μL containing 5 μg of membrane protein, testcompound ranging from 10 pM-20 μM), 1 μM GDP, and 10 nM GTP-Eu dilutedin 10 mM MgCl₂, 50 mM NaCl, and 0.0125% BSA, (final assayconcentrations). A DAMGO (Tyr-D-Ala-Gly-(methyl)Phe-Gly-ol)concentration-response curve (ranging from 12.8 pM-1 μM) was included onevery plate.

Assay plates were prepared immediately prior to assay following theaddition of 25 μL of Assay Buffer, 25 μL of test compound, and 25 μL GDPand GTP-Eu. The assay was initiated by the addition of 25 μL membraneprotein and allowed to incubate for 30 minutes. The assay plates werethen filtered with a Waters vacuum manifold connected to the housevacuum regulated to 10-12 in. Hg and washed with room temperature GTPWash Solution (2×300 mL). The bottoms of the plates were blotted toremove excess liquid. The plates were then immediately read to determinethe amount of bound GTP-Eu by measuring Time Resolved Fluorescence (TRF)on a Packard Fusion Plate ReaderVehicle: DMSO not to exceed 1% finalassay concentration.

The amount of bound GTP-Eu is proportional to the degree of activationof the mu opioid receptors by the test compound. The intrinsic activity(IA), expressed as a percentage, was determined as the ratio of theamount of bound GTP-Eu observed for activation by the test compound tothe amount observed for activation by DAMGO which is presumed to be afull agonist (IA=100). The final compounds of Examples 1 to 31demonstrated intrinsic activities of less than about 25. For example,the compounds of Examples 1, 11, and 24 had IA values in the GTP-Euassay of 0, −6, and 13, respectively. Thus, the compounds of the presentinvention have been shown to act as antagonists at the human mu opioidreceptor.

Assay 3: Rat Model of In Vivo Efficacy

In this assay the efficacy of test compounds was evaluated in a model ofgastrointestinal transit, which evaluates peripheral activity. Thisstudy was approved by the Institutional Animal Care and Use Committee atTheravance, Inc. and conformed to the Guide for the Care and Use ofLaboratory Animals published by the National Academy of Sciences(©1996).

a. Rat Gastric Emptying Assay

Test compounds were evaluated in the rat gastric emptying assay todetermine their ability to reverse loperamide-induced delayed gastricemptying. Rats were fasted up overnight prior to administration of testcompounds or vehicle by intravenous, subcutaneous, intramuscular or oralroutes of administration at doses ranging from 0.001 to about 30milligrams/kilogram (mg/kg). The administration of test compound wasfollowed by subcutaneous administration of loperamide at a dose of 1mg/kg or vehicle. Five minutes post loperamide or vehicleadministration, a non-nutritive, non-absorbable charcoal meal wasadministered via oral gavage and animals were allowed free access towater for the sixty minute duration of the experiment. Animals were theneuthanized via carbon dioxide asphyxiation followed by thoracotomy andthe stomach was carefully excised. The stomach was ligated at the loweresophageal sphincter and the pyloric sphincter to prevent additionalemptying during tissue removal. Gastric weight was then determined afterremoval of the ligatures.

b. Data Analysis and Results

Data was analyzed using the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.). Percent reversal curves wereconstructed by non-linear regression analysis using the sigmoidal doseresponse (variable slope) model and best-fit ID₅₀ values werecalculated. Curve minima and maxima were fixed to loperamide controlvalues (indicating 0% reversal) and vehicle controls (indicating 100%reversal), respectively. Results are expressed as ID₅₀, the doserequired for 50% reversal of the effects of loperamide, in milligramsper kilogram. The compound of Example 1 administered orally, exhibitedan ID₅₀ value of 0.48 mg/kg in the gastric emptying model.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1-20. (canceled)
 21. A method of treating a mammal having a medicalcondition ameliorated by treatment with a mu opioid receptor antagonistwherein the medical condition is opioid-induced bowel dysfunction orpost-operative ileus, the method comprising administering to the mammala therapeutically effective amount of a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and a compound offormula (I):

wherein R¹ is —OR^(a) or —C(O)NR^(b)R^(c); R², R³, and R⁴ are eachindependently C₁₋₃alkyl; R⁵ and R⁶ are each independently selected fromhydrogen, benzyl, and phenyl, wherein phenyl is optionally substitutedwith halo, or R⁵ and R⁶ together with the carbon atom to which they areattached form a cyclopentyl or cyclohexyl ring, R⁷ is selected fromhydroxy, C₁₋₃alkoxy, and —NR⁸R⁹; R⁸ and R⁹ are each independentlyselected from hydrogen, C₁₋₆alkyl, cyclohexyl, and benzyl, or R⁸ and R⁹together with the nitrogen atom to which they are attached formpiperidinyl or piperazinyl, wherein piperidinyl and piperazinyl areoptionally substituted with methyl; R^(a), R^(b), and R^(c) are eachindependently hydrogen or C₁₋₃alkyl; n is 0, 1, 2, 3, or 4; and m is 0or 1; wherein the substituents at the chiral centers marked by asterisksare in the trans configuration; provided that when n+m=1 and R⁷ ishydroxy or C₁₋₃alkoxy, then R⁵ and R⁶ are each independently selectedfrom benzyl and phenyl or R⁵ and R⁶ together with the carbon atom towhich they are attached form a cyclohexyl ring; or apharmaceutically-acceptable salt thereof.
 22. The method of claim 21wherein R¹ is —C(O)NH₂.
 23. The method of claim 21 wherein R⁵ and R⁶ areeach independently selected from hydrogen, benzyl, and phenyl, or R⁵ andR⁶ together with the carbon atom to which they are attached form acyclohexyl ring.
 24. The method of claim 21 wherein R⁷ is —NR⁸R⁹. 25.The method of claim 24 wherein R⁸ and R⁹ are each independently selectedfrom hydrogen, C₁₋₆alkyl, and benzyl or R⁸ and R⁹ together with thenitrogen atom to which they are attached form piperidinyl.
 26. Themethod of claim 25 wherein R¹ is —OH or —C(O)NH₂; R² and R³ are eachmethyl or R² and R³ are each ethyl; R⁴ is methyl; R⁵ and R⁶ are eachhydrogen; and n is 0 and m is
 0. 27. The method of claim 21 wherein themedical condition is opioid-induced bowel dysfunction.
 28. The method ofclaim 21 wherein the medical condition is post-operative ileus.
 29. Amethod of enhancing motility of the gastrointestinal tract in a mammal,the method comprising administering to the mammal a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier and acompound of formula (I):

wherein R¹ is —OR^(a) or —C(O)NR^(b)R^(c); R², R³, and R⁴ are eachindependently C₁₋₃alkyl; R⁵ and R⁶ are each independently selected fromhydrogen, benzyl, and phenyl, wherein phenyl is optionally substitutedwith halo, or R⁵ and R⁶ together with the carbon atom to which they areattached form a cyclopentyl or cyclohexyl ring, R⁷ is selected fromhydroxy, C₁₋₃alkoxy, and —NR⁸R⁹; R⁸ and R⁹ are each independentlyselected from hydrogen, C₁₋₆alkyl, cyclohexyl, and benzyl, or R⁸ and R⁹together with the nitrogen atom to which they are attached formpiperidinyl or piperazinyl, wherein piperidinyl and piperazinyl areoptionally substituted with methyl; R^(a), R^(b), and R^(c) are eachindependently hydrogen or C₁₋₃alkyl; n is 0, 1, 2, 3, or 4; and m is 0or 1; wherein the substituents at the chiral centers marked by asterisksare in the trans configuration; provided that when n+m=1 and R⁷ ishydroxy or C₁₋₃alkoxy, then R⁵ and R⁶ are each independently selectedfrom benzyl and phenyl or R⁵ and R⁶ together with the carbon atom towhich they are attached form a cyclohexyl ring; or apharmaceutically-acceptable salt thereof.
 30. The method of claim 29wherein R⁵ and R⁶ are each independently selected from hydrogen, benzyl,and phenyl, or R⁵ and R⁶ together with the carbon atom to which they areattached form a cyclohexyl ring.
 31. The method of claim 29 wherein R⁷is —NR⁸R⁹.
 32. The method of claim 31 wherein R⁸ and R⁹ are eachindependently selected from hydrogen, C₁₋₆alkyl, and benzyl or R⁸ and R⁹together with the nitrogen atom to which they are attached formpiperidinyl.
 33. The method of claim 32 wherein R¹ is —OH or —C(O)NH₂;R² and R³ are each methyl or R² and R³ are each ethyl; R⁴ is methyl; R⁵and R⁶ are each hydrogen; and n is 0 and m is
 0. 34. A method ofantagonizing a mu opioid receptor in a mammal, the method comprisingadministering to the mammal a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of formula (I):

wherein R¹ is —OR^(a) or —C(O)NR^(b)R^(c); R², R³, and R⁴ are eachindependently C₁₋₃alkyl; R⁵ and R⁶ are each independently selected fromhydrogen, benzyl, and phenyl, wherein phenyl is optionally substitutedwith halo, or R⁵ and R⁶ together with the carbon atom to which they areattached form a cyclopentyl or cyclohexyl ring, R⁷ is selected fromhydroxy, C₁₋₃alkoxy, and —NR⁸R⁹; R⁸ and R⁹ are each independentlyselected from hydrogen, C₁₋₆alkyl, cyclohexyl, and benzyl, or R⁸ and R⁹together with the nitrogen atom to which they are attached formpiperidinyl or piperazinyl, wherein piperidinyl and piperazinyl areoptionally substituted with methyl; R^(a), R^(b), and R^(c) are eachindependently hydrogen or C₁₋₃alkyl; n is 0, 1, 2, 3, or 4; and m is 0or 1; wherein the substituents at the chiral centers marked by asterisksare in the trans configuration; provided that when n+m=1 and R⁷ ishydroxy or C₁₋₃alkoxy, then R⁵ and R⁶ are each independently selectedfrom benzyl and phenyl or R⁵ and R⁶ together with the carbon atom towhich they are attached form a cyclohexyl ring; or apharmaceutically-acceptable salt thereof.